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Latest revision as of 03:40, 18 October 2014

Protocols

1 Media, Plates, and Solutions
2 Basic Cloning
3 Escherichia coli Protocols (XL1-Blue and XL10-Gold)
4 Saccharomyces cerevisiae (PyE1 Yeast)
5 Flow Cytometry
- 5.1 Dilutions
- 5.2 Preparations for Analysis using C6 Accuri Flow Cytometer
6 Fluorescence-Activated Cell Sorting
7 Protein Expression
8 Stability Analysis
- 8.1 Circular Dichroism: Wavelength Scan
- 8.2 Circular Dichroism: Guanidine Melt

Media, Plates, and Solutions

Competent Cell Media Buffer (CCMB)

Mix the following to a 2 L container:
- 100 g glycerol (liquid)
- 10 mL x 1 M potassium acetate
- 11.8 g CaCl2*H2O
- 4 g MnCl2
- 2 g MgCl2
- 1 L of dH2O

Sterile filter or autoclave in a 1 L bottle

Super Optimal Broth (SOB)

Mix the following to a 2 L container:
- 20 g tryptone
- 5 g yeast extract
- 10 mL x 1 M NaCl
- 2.5 mL x 1 M KCl
- 1 L of dH2O

Sterile filter or autoclave in a 1 L bottle

Phosphate Buffered Saline (PBS) Solution

Mix the following in a 2 L container or 1 L beaker:
- 8 g NaCl
- 1.44 g Na2HPO4
- 0.8 g KCl
- 0.24 g KH2PO4
- 1 L of dH2O
Buffer to pH 7.4

Sterile filter or autoclave in a 1 L bottle

PBSF (PBS for Flow)

Mix the following in a 1 L beaker:
- 25 mL 20X PBS, pH 7.4
- 475 mL H2O
- 2.5 g BSA (0.5%)*

Sterile filter in a 1 L bottle and store at 4 °C

Yeast Extract Peptone Dextrose (YPD)

Mix the following into 950 mL of dH2O in a 1 L bottle:
- 20 g peptone
- 10 g yeast extract

Autoclave
Add 50 mL 40% glucose
Sterile filter into a 1 L bottle

Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures.
Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving.

Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His)

Synthesized by the Yeast Resource Center at the University of Washington's Department of Genome Sciences and Department of Biochemistry.

Guanidinium Hydrogen Chloride

For maximum effectiveness, final concentration should be approximately 8.5 M in PBS
Add the following to a 500 mL beaker and mix:

- 203 g guanidinium hydrogen chloride
- 250 mL PBS solution*
- Add dilute HCl to pH 7.4

*It is not necessary to filter or autoclave.
*Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary.

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Basic Cloning

Polymerase Chain Reaction

All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation.
Mix the following in a 0.2 mL microcentrifuge tube on ice:
25 μL GoTaq® Green Master Mix 2X
1-5 μL of 10 μM forward primer
1-5 μL of 10 μM reverse primer
<250 ng of DNA template
QS 50 μl nuclease-free H2O
Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.

Error-prone Polymerase Chain Reaction

Prepare 50 μL reaction:
5 μL 10X Mutazyme II Rxn Buffer
1 μL 40 mM dNTP mix (200 μM each final)
1 μL 20 μM forward primer
1 μL 20 μM reverse primer
1 μL Mutazyme II DNA polymerase (2.5 U/μL)
0.01 ng template
QS 50 μL diH2O

Program thermocycler as follows:
95 °C, 2 min
95 °C, 30 sec
XX °C*, 30 sec
72 °C, X min**
32 cycles
72 °C, 10 min
4 °C, hold

*Adjust annealing temperature according to Tm of primer.
**Adjust extension time according to the length of amplified DNA.

Note: Use 0.01 ng of template (calculate by insert and not by total plasmid).
Calculate amount of template to use as follows:
(bp for amplified region) / (bp in total plasmid) = % amplified region
(conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region
Note: Never pipette less than 0.5 μL.
(0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR

Restriction Endonuclease Reaction (Digestion)

All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc.
Mix the following in a 0.2 mL PCR tube:
1 μg of DNA
5 μL of the appropriate 10X New England Biolab® Buffer
1 μL of each restriction enzyme (add last)
QS 50μL nuclease-free H2O
Incubate the reaction for 1 hr
Heat inactive the reaction at the appropriate temperature

Note: Thaw the restriction enzyme(s) on ice to improve shelf life.

Ligation

T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc.
1. Prepare the following in a 0.2 mL microcentrifuge tube:
50.0 ng vector DNA*
37.5 ng vector DNA*
2 μL 10X T4 DNA Ligase Buffer
1 μL T4 DNA Ligase
QS 20 μL diH2O
2. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight.
3. Heat inactivate at 65 °C for 10 minutes.
4. Chill on ice before starting a transformation reaction.

*The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at: http://nebiocalculator.neb.com/#!/

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Escherichia coli Protocols (XL1-Blue and XL10-Gold)

Chemically Competent Cell Cultures

Competent cells take two days to culture and aliquot.
Day 1:
1. Streak an aliquot of competent cells onto two LB-plates without antibiotics.*
2. Incubate at 37 °C overnight.
Day 2:
1. In two 250 mL baffle flasks add 50 mL of SOB media.
2. Scrape as many single colonies into either flask.
3. Incubate and shake at 37 °C and 250 rpm for 2-3 hours.
4. Check the optical density of the cells at 600 nm after 2 hours.
5. Stop incubation when cultures reach approximately 0.5 optical density.
6. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes.
7. Spin down the cells at 2500 rpm at 4 °C for 15 minutes.
8. Decant the supernatant.
9. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing.
10. Incubate the cells on ice for 20 minutes.
11. Spin down the cells at 2500 rpm at 4 °C for 10 minutes.
12. Decant the supernatant.
13. Resuspend the cells in 4 mL of CCMB.
14. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.**
15. Store the competent cell aliquots at -80 °C.

*Streak in such a way that there should be individual colony growth and no clumps after the incubation.
**We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time.

Note: After removing the cells from incubation keep them on ice or as cold as possible.

Chemically Competent Cell Transformations

1. Thaw competent E. coli cells on ice (XL1-Blue or XL10-Gold).*
2. Add 50 μL of competent cells to sterile 14 mL culture tube.
3. Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube.
4. Equilibrate the cells on ice for 10 minutes.
5. Heat shock the cells at 42 °C for 30-45 seconds.**
6. Immediately place the cells back on ice for 3 minutes.
7. Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes.
8. Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate.
9. Invert the plate and incubate at 37 °C overnight.

*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells.
**Do not heat shock for an extended duration as this may damage and/or kill your cells.

Overnights

1. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s).
2. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip.
3. Swirl the colony tip in the tube, there should be no visible cell clumps.
4. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours.

DNA Extraction and Mini-Preps

All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.

Glycerol Stocks

1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube.
2. Spin down the culture at 3000 rpm for 3 minutes.
3. Decant the supernatant.
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water.
5. Transfer the resuspension to a cryogenic vial.
6. Store the glycerol stock at -80 °C.

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Saccharomyces cerevisiae (PyE1 Yeast)

Chemically Competent Cell Cultures

This process take four days in lab with a one day wait for incubation.
Day 1:
1. Streak yeast cells onto a YPD plate.*
2. Invert the plate and incubate at 30 °C for 2 days.
Day 3:
1. Add 50 mL of YPD liquid media into a 250 mL baffle flask.
2. Swipe as many individual colonies as you can see into the YPD media.**
3. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours.
Day 4:
1. Take an optical density measurement.
2. In three 250 mL baffle flask add the portions of the overnight liquid culture.
3. Dilute each culture to approximately 0.4 optical density with YPD.
4. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6.
5. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes.
6. Spin down the cells at 4000 x g for 5 minutes at 4 °C.
7. Decant the supernatant.
8. Resuspend the cells in 100 mL total for all three culture of dH2O.
9. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes.
10. Spin down the cells as above.
11. Decant the supernatant.
12. Resuspend each in 3 mL of 100 mM lithium acetate.
13. Transfer both cultures into a single 15 mL conical centrifuge tube.
14. Spin down the cells at 3000 rpm for 5 minutes.
15. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL.
16. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol.
17. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.***

*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies.
**Collect only individual visible colonies. Do not collect clumps or satellite colonies.
***The volume of aliquots depends on the number to transformations you intend to do at a time.

Chemically Competent Transformations

This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations.

1. Add the following to 50 μL of yeast competent cells:
240 μL of polyethylene glycol - 3350 (PEG-3350)
36 μL of 1 M lithium acetate
32 μL of milliQ H2O
2. Mix the mixture by gently pipetting or vortexing.
3. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube.
4. Add 1 μL (~100-200 ng) of DNA.*
5. Mix the mixture by gentle pipetting or vortexing.
6. Incubate the mixture at 30 °C for 30 minutes.
7. Heat shock the mixture at 42 °C for 20 minutes.
8. Spin down the cells in a microcentrifuge for ~1 minute.
9. Decant the supernatant.
10. Resuspend the cell pellets in 200 μL of dH2O.
11. Spin down the cells in a microcentrifuge for ~1 minute.
12. Resuspend the cell pellets in 200 μL of dH2O.
13. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate.
14. Invert and incubate at 30 °C for 2 days.

*The exact amount of DNA depends on the transformation efficiency of your competent cells.

Overnight Culturing

1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose.
2. Swipe 3 isolated yeast colonies and add them to the culture tube media.
3. Incubate and shake at 37 °C at 250 rpm for 2 days.

Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.

Culture Passaging

1. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose.
2. Take 20-50 μL from a previous overnight or passage culture and add it to the culture media.
3. Incubate and shake at 37 °C at 250 rpm for 2 days.

Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.
Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least one living cell is passaged.

Glycerol Stocks

1. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube.
2. Spin down the culture at 3000 rpm for 3 minutes.
3. Decant the supernatant.
4. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water.
5. Transfer the resuspension to a cryogenic vial.
6. Store the glycerol stock at -80 °C.

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Flow Cytometry

Dilutions

1. From an overnight culture measure the optical density at 660 nm by making 1:10 dilutions.
2. Take enough culture to make a 1 mL aliquot with an OD of 0.4.
3. Spin down the aliquot in a 1.5 mL centrifuge tube at 3000 rpm for 3 minutes.
4. Decant the supernatant.
5. Resuspend the cell pellet in 800 μL of the appropriate selective dropout media and 200 μL of 20% glucose.
6. Transfer the new culture to a 14 mL culture tube.
7. Incubate and shake at 30 °C and 250 rpm for at least 6 hours (1.2-1.6 optical density).

Preparations for Analysis using C6 Accuri Flow Cytometer

1. From the dilution previously made, measure the optical density, roughly 1.2-1.6.
2. Make an aliquot of 500 μL of the dilution culture in a 1.5 mL centrifuge tube.
3. Spin down the aliquot at 3000 rpm for 3 minutes.
4. Decant the supernatant.
5. Resuspend the cell pellet in 500 μL of PBSF.
6. Spin down the resuspension at 3000 rpm for 3 minutes.
7. Decant the supernatant.
8. Resuspend the cell pellet in another 500 μL of PBSF.*
9. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a diH2O cycle.
10. Load the sample onto the sip.
11. Run the sample with 100,000 cell count.
12. Repeat for all samples and make sure to change data cells otherwise the old data will be erased.
13. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a diH2O cycle.

*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.

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Fluorescence-Activated Cell Sorting

Dilution of Cells:

1. Take OD600 of the cultures.
2. Calculate volume to spin down for OD of 0.4 in appropriate volume (typically 1 mL) - C1V1=C2V2
3. Spin down appropriate volume in eppendorf tubes for 3 min at 3000 rpm.
4. Aspirate off supernatant.
5. Resuspend pellet in 1 mL C-Ura + 2% glucose (or other appropriate media).
6. Shake in 14 mL culture tube at 30 °C for 6 hrs.

Sample Prep:

1. Transfer 500 μL of samples and negative control to eppendorf tubes.
2. Spin down cells (3000 rpm, 3 min).
3. Aspirate off supernatant. Resuspend in PBSF.
4. Spin down cells (3000 rpm, 3 min).
5. Aspirate off supernatant. Resuspend in PBSF.

Using the Fluorescence-Activated Cell Sorter

1. Load the “iGEM Template” file in the FACS Software.
2. Make sure the stream is stable.
3. Run all controls and record 100,000 events for analysis.
4. While running controls, set FSC/SSC and FSC-H/FSC-W gates.
5. Run library and record 100,000 events for analysis.
6. Set gate for top 1.00% of GFP fluorescence.
7. Sort cells falling in all three gates. Sort ten-fold over library size.
8. Run bleach and diH2O through FACS to avoid cross-contamination.

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Protein Expression

Overnight Cultures

1. Add 25 mL TB and 25 μL 1000X Kan to a 250 mL baffled flask.
2. Stab a glycerol stock with a P1000 pipette and swirl in the flask of media.
3. Put flask in 37 °C shaker at 250 rpm for 16-20 hrs.

Protein Expression

1. Add 500 μL 1000X Kanamycin and 1 mL MgSO4 to 500 mL TB in 2 L baffled flask.
2. Transfer 10 mL overnight culture to TB.
3. Shake at 37 °C and 250 rpm until OD600 is between 0.5 and 0.8.
4. Allow flask to rest at room temp for 30 min.
5. Add 125 μL 1 M IPTG.
6. Shake flask at 18 °C for ~16-20 hrs.

Protein Extraction and Purification

1. Transfer cell culture to centrifuge tube.
2. Centrifuge culture at 4000 x g for 10 min.
3. Discard supernatant.
4. Resuspend pellet in 25 mL wash buffer and add 250 μL of 100X PMSF, 250 μL of 100 mg/mL lysozyme, and 250 μL of 10 mg/mL DNAse.
5. Sonicate sample with 0.25 inch probe for 5 min at 70% amplitude with 20 sec on and off pulses.
6. Take 50 μL total sample.
7. Transfer lysate to SS-34 centrifuge tube.
8. Centrifuge for 30 min at 18000 x g.
9. Take 50 μL soluble sample.

Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography)

1. Add 5 mL 50%(v/v) nickel resin in ethanol to a 25 mL gravity flow column and allow to settle to 2.5 mL(CV).
2. Rinse with 10CV dH2O.
3. Equilibrate with 10CV lysis buffer.
4. Load sample onto column.
5. Wash column with 15CV lysis buffer.
6. Perform 2 additional wash steps with 15CV.
7. Elute sample in 10CV elution buffer and collect eluate.
8. Take 50 μL pure sample.

Size Exclusion Chromatography (SEC)

1. Concentrate sample to as high as possible without inducing protein aggregation.
2. Pre-equilibrate Superdex 75 column with 48 mL PBS.
3. Inject 500 μL sample onto column.
4. Run 36 mL PBS through column at 0.5 mL/min, collecting 1 mL fractions.
5. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 mL).
6. Pool fractions containing protein.

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Stability Analysis

Circular Dichroism: Wavelength Scan

1. Load 1mm cuvette with 400 μL protein solution onto CD.
2. Take wavelength scan:
260 nm-190 nm
sample every 1 nm
averaging time 3 sec
1 scan
step scan
25 °C
3. Record wavelength which gives strongest signal (222 nm).

Circular Dichroism: Guanidine Melt

1. Load 1 cm cuvette containing 1.996 mL of 0.05 mg/mL protein solution and stirrer onto CD.
2. Prepare 8 mL of 0.05 mg/mL protein in concentrated guanidine solution.
3. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe.
4. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval.
5. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing.

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+<body>
+<h1> <center>Protocols</center> </h1>
-	<head>
-		<style>
+<html> <a name="Media, Buffers and Solutions"></a> </html>
- 			 body  {background-color:white}
-  				h1	{color:purple ;
+    <h2> Media, Plates, and Solutions </h2>
-					font-family:'Lucida Sans Unicode', 'Lucida Grande', sans-serif" ;
-					font-size:250%}
-  				h2	{color:purple;
-					font-family:'Lucida Sans Unicode', 'Lucida Grande', sans-serif" }
-  				h3	{color:purple;
-					font-family:'Lucida Sans Unicode', 'Lucida Grande', sans-serif" }
-  				p	{color:#39275B;
-					font-family:'Lucida Sans Unicode', 'Lucida Grande', sans-serif"; align = left }
-		</style>
+        <h3> Competent Cell Media Buffer (CCMB) </h3>
+            <p>
+                                Mix the following to a 2 L container:<br>
+- 100 g glycerol (liquid) <br>
-	</head>
+                          - 10 mL x 1 M potassium acetate <br>
-<a name="linktotop">Back To Top</a>
-</html>
-<h1> Protocols </h1>
+                          - 11.8 g CaCl2*H2O <br>
-	<h2> Media, Plates and Solutions </h2>
+                          - 4 g MnCl2 <br>
-		<h3> 40% and 20% Glucose </h3>
+                          - 2 g MgCl2 <br>
-			<p>
+                          - 1 L of dH2O <br>
-g for 40% or 20g for 20% of Glucose <br>
+<br>
-				Mix in 100mL diH2O <br>
-				Sterile filter into a 150mL bottle <br>
+                           Sterile filter or autoclave in a 1 L bottle
-			</p>
-		<h3> 20% Glycerol </h3>
+        </p>
-			<p>
-g Glycerol (Liquid) <br>
-				Mix in 100mL diH2O <br>
-				Sterile filter into a 150mL bottle <br>
-			</p>
-		<h3> Competent Cell Media Buffer (CCMB) </h3>
+        <h3> Super Optimal Broth (SOB) </h3>
+                        <p>
+                                Mix the following to a 2 L container: <br>
-			<p>
-g Glycerol (liquid) <br>
-mL x 1M Potassium Acetate <br>
-.8g CaCl2*H2O <br>
-g MnCl2 <br>
-g MgCl2 <br>
-     				Mix in 1L diH2O <br>
-     				Sterile filter or autoclave (20min at 121C and 20psi) in a 1L bottle
-			</p>
-		<h3> Luria Broth (LB) </h3>
+                          - 20 g tryptone <br>
-			<p>
+                         - 5 g yeast extract <br>
-g tryptone <br>
-g yeast extract <br>
-g NaCl <br>
-mL diH2O <br>
-				Autoclave in two 500 ml bottle (20 min at 121C and 20psi)
-                               *If using antibiotics create a separate aliquot
+                         - 10 mL x 1 M NaCl <br>
-			</p>
-		<h3> LB-Agar </h3>
+                          - 2.5 mL x 1 M KCl <br>
-			<p>
+                          - 1 L of dH2O <br>
-ml LB as above <br>
-g agar <br>
+                       <br>
-L diH2O <br>
-				Autoclave in two 500mL bottles (20 min at 121C at 20psi) <br>
-                               *If using antibiotics create a separate aliquot
-			</p>
-		<h3> Super Optimal Broth (SOB) </h3>
+                      Sterile filter or autoclave in a 1 L bottle
-                        <p>
-g BactoTryptone <br>
-g BactoYeast Extract <br>
-mL x 1M NaCl <br>
-.5mL x 1M KCl <br>
-     				Mix in 1L of diH2O <br>
-     				Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle
                          </p>
-		<h3> Phosphate Buffered Saline (PBS) Solution </h3>
-			<p>
-g NaCl <br>
-.44g Na_2HPO_4 <br>
-.8g KCl <br>
-.24g KH_2PO_4 <br>
-				Mix in 1L of diH2O and buffer to pH 7.4 <br>
-				Sterile filter or autoclave (20 min at 121C and 20psi) in a 1L bottle
-			</p>
-		<h3> Tryptone Phosphate Buffer (TB) </h3>
+        <h3> Phosphate Buffered Saline (PBS) Solution </h3>
+        <p>
+                                Mix the following in a 2 L container or 1 L beaker: <br>
-			<p>
-				Mix the following in a 1L bottle: <br>
-g Tryptone <br>
-g Yeast Extract <br>
-mL Glycerol <br>
-mL of diH2O <br>
-				Autoclave (liquid cycle LOOK UP SPECIFICATIONS) <br>
-				Cool and add 5mL of 100X Potassium Phosphate Salts (17mM KH2PO4 and 72mM K2HPO4)
-			</p>
-		<h3> Yeast Extract Peptone Dextrose (YPD) </h3>
+- 8 g NaCl <br>
-			<p>
+                - 1.44 g Na2HPO4 <br>
-g Bacto Peptone
-g Yeast Extract
-				Mix into 950mL of diH2O in a 1L bottle
-				Autoclave (20min at 121C at 20psi)
-				Add 50mL 40% Glucose
-				Sterile Filter into a 1L bottle
-				For long-term liquid media storage, do not add 40% Glucose instead add the glucose directly into cell cultures.
-				For YPD-plates add 24g Bacto Agar to the Bacto Peptone and Yeast Extract before autoclaving.
-			</p>
-		<h3> Selective Dropout media, C-Uracil and C-Histidine (C-Ura and C-His) </h3>
+                - 0.8 g KCl <br>
-			<p>
+                - 0.24 g KH2PO4 <br>
-				Synethesized by the Yeast Resource Center at the Univeristy of Washington's
-					Department of Genome Sciences and Department of Biochemistry.
-			</p>
-		<h3> Guanidinium Hydrogen Chloride </h3>
+                - 1 L of dH2O <br>
-			<p>
+                                 Buffer to pH 7.4 <br><br>
-				For maximum effectiveness, final concentration should be 8.5M in PBS <br>
-g Guanidinium Hyrdogen Chlordie <br>
-mL PBS solution <br>
-				Add dilute HCl to 7.4pH <br>
-				*Alternatively add slightly less than 250mL of PBS in order to buffer the solution to the appropriate volume.
+                             Sterile filter or autoclave in a 1 L bottle
-			</p>
-	<h2> Basic Cloning </h2>
+        </p>
-		<h3> Polymerase Chain Reactions </h3>
-			<p>
-				All PCRs were done using a standard 50uL reaction volume. <br>
-				PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation. <br>
-				Protocols for the PROMEGA GoTaq Green Master Mix 2X: <br>
-				Mix the following in a 0.2mL PCR tube on ice: <br>
-uL GoTaq® Green Master Mix 2X <br>
--5uL of 10uM Forward primer <br>
--5uL of 10uM Reverse primer <br>
-					<250ng of DNA template <br>
-					Nuclease-Free Water to 50μl <br>
-				In a thermocyler conduct the reaction...
-			</p>
-		<h3> Error-prone Polymerase Chain Reaction </h3>
-			<p>
+                       <h3>PBSF (PBS for Flow)</h3>
+<p>
-				Prepare 50uL reaction: <br>
-uL 10X Mutazyme II Rxn Buffer <br>
-uL 40mM dNTP mix (200uM each final) <br>
-uL 20uM forward primer <br>
-uL 20uM reverse primer <br>
-uL Mutazyme II DNA polymerase (2.5U/uL) <br>
-.01ng template <br>
-				QS 50uL diH2O <br>
-				<br>
-				Thermocycler: <br>
-C, 2min <br>
-C, 30sec <br>
-				XXC*, 30sec <br>
-C, Xmin** <br>
-cycles <br>
-C, 10min <br>
-C, hold
-				<br>
-				*Adjust annealing temperature according to Tm of primer. <br>
-				**Adjust extension time according to the length of amplified DNA. <br>
-				<br>
-				Note: Use 0.01ng (calculate by insert and not by total plasmid). <br>
-				Calculate amount of template to use. <br>
-				(bp for amplified region) / (bp in total plasmid) = % amplified region <br>
-				(conc of total plasmid) x (% amplified region as a decimal) = conc of amplified region <br>
-				<br>
-				Note: Will probably need to dilute. Never pipette less than 0.5uL. <br>
-				(0.01ng) / (conc of amplified region) = vol to add to PCR  <br>
-			</p>
+           Mix the following in a 1 L beaker:
+<br>
-		<h3> Restriction Endonuclease Reaction (Digestion) </h3>
+  - 25 mL 20X PBS, pH 7.4<br>
-			<p>
-				All restriction enzyme reactions were done using a 50ul reaction volume. <br>
-				Restriction enzymes and buffers were purchased from New England Biolabs Incorporated. <br>
-				Protocols for various New England Biolab restriction enzyme reactions: <br>
-				Mix the following in a 0.2mL PCR tube: <br>
-uL of each Restriction Enzyme, add the RE last <br>
-ug of DNA <br>
-uL of the appropriate 10X New Englan Biolab Buffer <br>
-					Nuclease-Free Water to 50uL <br>
-				Incubate the reaction for 1hr <br>
-				Heat inactive the the reaction at the appropriate temperature <br>
-				Notes:  Add the restriction enzyme(s) to the reaction last <br>
+  - 475 mL H2O<br>
-					Thaw the restriction enzyme(s) on ice to improve shelf life <br>
-			</p>
-		<h3> Ligation </h3>
+  - 2.5 g BSA (0.5%)*
+<br><br>
-			<p>
+Sterile filter in a 1 L bottle and store at 4 °C
-				T4 DNA Ligase and Buffer was purchased from New England BioLabs Corporation. <br>
-. Prepare the following in a 0.2mL mircocentrifuge tube: <br>
-.0ng Vector DNA* <br>
-.5ng Vector DNA* <br>
-uL 10X T4 DNA Ligase Buffer <br>
-uL T4 DNA Ligase <br>
-					Add diH2O to 20uL <br>
-. Incubate the reaction at room temperature for 10-30 minutes or at 16C overnight. <br>
-. Heat inactivate at 65C for 10 minutes. <br>
-. Chill on ice before starting a transformation reaction. <br>
-				* The exact amount of DNA is dependant on the number of base pairs. In order to conduct a proper reaction consult the New Englan Biolab Ligation Calculator at:
-					http://nebiocalculator.neb.com/#!/
-			</p>
-	<h2> <i> Escherichia coli Protocols (XL1-Blue and XL10-Gold) </i> </h2>
+</p>
-		<h3> Chemically Competent Cell Culturing </h3>
+        <h3> Yeast Extract Peptone Dextrose (YPD) </h3>
+<p>
-			<p>
-			Competent Cells take two days to culture and aliquot. <br>
-				Day 1: <br>
-. Streak an aliquot of Compentent Cells onto two LB-plates without anti-biotics.* <br>
-. Incubate at 37C overnight. <br>
-				Day 2: <br>
-. In two 250mL baffle flask add 50mL of S.O.B. media. <br>
-. Scrape as many single colonies into either flask. <br>
-. Incubate and shake at 37C and 250rpm for 2-3 hours. <br>
-. Check the optical density of the cells at 550nm after 2 hours. <br>
-. Stop incubation when cultures reach approximately 0.5 optical density. <br>
-. Add the contents of the flask into separate 50mL flat bottomed centrifuge tubes. <br>
-. Spin down the cells at 2500rpm at 4C for 15 minutes. <br>
-. Decant the supernatant. <br>
-. Resuspend the cells in 16mL of CCMB by pipetting or gentle vortexing. <br>
-. Incubate the cells on ice for 20 minutes. <br>
-. Spin down the cells at 2500rpm at 4C for 10 minutes. <br>
-. Decant the supernatant. <br>
-. Resuspend the cells in 4mL of CCMB. <br>
-. Quickly aliquot the cells into 1.7mL cryogenic vials or 1.5mL centrifuge tubes.** <br>
-. Store the competent cell aliquots at -80C. <br>
-					*Streak in such a way that there should be invidual colony growth and no clumps after the incubation. <br>
-					**We did this in a -20C cold room and using a automated repeater pipette. <br>
-					**The volume of each aliquot depends on the number of transformations you intend to do at a time. <br>
-					***After removing the cells from incubation keep them on ice or as cold as possible. <br>
-			</p>
-		<h3> Chemically Competent Cell Transformations </h3>
-			<p>
+                                   Mix the following into 950 mL of dH2O in a 1 L bottle:
-.   Thaw competent <i> E.coli </i> cells on ice (XL1-Blue or XL10-Gold)* <br>
+<br>
-.   Add 50 uL of competent cells to sterile 15 mL conical centrifuge tubes <br>
+                - 20 g peptone <br>
-.   Add 1uL (~100-200ng)* of the miniprep to each culture tube <br>
-.   Equilibrate the cells on ice for 10 min <br>
-.   Heat shock the cells at 42C for 30-45 seconds** <br>
-.   Immediately place the cells back on ice for 3 min <br>
-.   Add 250 uL LB media without antibiotics and shake at 250 rpm and 37C for 30 min <br>
-.   Spread 10ul and 290uL on an appropriate LB-antibiotic plate<br>
-.   Invert the plate and incubate at 37C overnight
-				*The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br>
+               - 10 g yeast extract
-				** Do not heat shock for an extended duration as this may damage and/or kill your cells.
-			</p>
-		<h3> Overnights </h3>
-			<p>
-. In a 14mL round-bottom tube, add 3-5mL of LB and an appropriate volume of antibiotic(s). <br>
-. Swipe several individual colonies, do not collect satelites or colony clumps, with a pipette tip. <br>
-. Swirl the colony tip in the tube, there should be no visible cell clumps. <br>
-. Incubate and shake the tube at 37C at 250rpm for 12-16 hours and no longer than 20 hours. <br>
-			</p>
-		<h3> DNA-Extraction and mini-preps </h3>
+<br><br>
-			<p>
+            Autoclave <br>
-				All DNA mini-preps were prepared using EPOCH minikits and following the supplied protocols.
-			</p>
-		<h3> Glycerol Stocks </h3>
+            Add 50 mL 40% glucose <br>
-			<p>
+            Sterile filter into a 1 L bottle <br>
-. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br>
-. Spin down the culture at 3000rpm for 3 minutes. <br>
-. Decant the supernatant. <br>
-. Resuspend the cells in 500uL of 40% Glycerol and 500uL of LB(no antibiotics) or water. <br>
-. Transfer the resuspension to a cryogenic vial. <br>
-. Store the glycerol stock at -80C. <br>
-			</p>
-	<h2> <i> Saccharomyces cerevisiae </i> (PYE1 Yeast) </h2>
+                    <br>
-		<h3> Chemically Competent Cell Culturing </h3>
+            Note: For long-term liquid media storage, do not add 40% glucose. Instead add the glucose directly into cell cultures. <br>
-			<p>
-				This process take 4 days in lab with a 1 day wait for incubation. <br>
-				Day 1: <br>
+            Note: For YPD-plates add 24 g agar to the peptone and yeast extract before autoclaving. <br>
-. Streak yeast cells onto a YPD plate.* <br>
-. Invert the plate and incubate at 30C for 2 days. <br>
-				Day 3: <br>
+            </p>
-. Add 50mL of YPD liquid media into a 250mL baffle flask. <br>
-. Swipe as many individual colonies as you can see into the YPD media.** <br>
-. Incubate and shake the culture at 30C at 250rpm overnight approximately 24 hours. <br>
-				Day 4: <br>
-. Take an optical density measurement. <br>
-. In three 250mL baffle flask add the portions of the overnight liquid culture. <br>
-. Dilute each culture to approximately 0.4 optical density with YPD. <br>
-. Incubate and shake the cultures at 30C at 250rpm until the optical density reaches 1.2-1.6. <br>
-. Collect each culture into separate 50ml flat-bottomed centrifuge tubes. <br>
-. Spin down the cells at 4000g for 5 minutes at 4C. <br>
-. Decant the supernatant. <br>
-. Resuspend the cells in 100mL total for all three culture of dH2O. <br>
-. Combine the suspensions into two 50mL flat-bottomed centrifuge tubes. <br>
-. Spin down the cells as above. <br>
-. Decant the supernatant. <br>
-. Resuspend each in 3mL of 100mM Lithium Acetate. <br>
-. Transfer both cultures into a single 15mL conical centrifuge tube. <br>
-. Spin down the cells at 3000rpm for 5 minutes. <br>
-. Resuspend the cells in 0.75mL of 100mM Lithium Acetate, total volume is roughly 2mL. <br>
-. Qualitatively bring up the volume to 3.5mL by adding 40% Glycerol. <br>
-. Aliquot the cells into 1.5mL centrifuge tubes or 1.7mL cryogenic vials.*** <br>
-				*Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br>
+        <h3> Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His) </h3>
-				**Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br>
+            <p>
-				***The volume of aliquots depends on the number to transformations you intend to do at a time. <br>
-			</p>
-		<h3> Chemically Competent Transformations </h3>
-			<p>
+                Synthesized by the Yeast Resource Center at the University of Washington's
-				This protocol assumes a 50uL aliquot of yeast competent cells were made. <br>
-				Furthermore, this protocol prepares enough cells for 6 yeast transformations. <br>
-				<br>
-. Add the following to 50uL of yeast competent cells: <br>
-uL of Polyethylene Glycol - 3350 (PEG-3350) <br>
-uL of 1M Lithium Acetate <br>
-uL of dH2O <br>
-. Mix the mixture by gentle pipetting or vortexing. <br>
-. Aliquot 59uL of the mixture into a 0.2mL microcentrifuge tube. <br>
-. Add 1uL (~100-200ng) of DNA. <br>
-. Mix the mixture by gentle pipetting or vortexing. <br>
-. Incubate the mixture at 30C for 30 minutes. <br>
-. Heat shick the mixture at 42C for 20 minutes. <br>
-. Spin down the cells in a microcentrifuge for ~1 minute. <br>
-. Decant the supernatant. <br>
-. Resuspend the cell pellets in 200uL of dH2O. <br>
-. Spin down the cells in a microcentrifuge for ~1 minute. <br>
-. Resuspend the cell pellets in 200uL of dH2O. <br>
-. Plate 50-150uL of the mixture onto an appropriate Selective Dropout Media plate. <br>
-. Invert and incubate at 30C for 2 days. <br>
-				*The exact amount of DNA depends on the transformation efficiency of your competent cells.
-			</p>
+                    Department of Genome Sciences and Department of Biochemistry.
-		<h3> Overnight Culturing </h3>
-			<p>
+            </p>
-. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br>
-. Swipe 3 invidually visible yeast colonies and add them to the culture tube media. <br>
-. Incubate and shake at 37C at 250rpm for 2 days. <br>
-				Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%.
-			</p>
-		<h3> Culture Passaging </h3>
+        <h3> Guanidinium Hydrogen Chloride </h3>
-			<p>
+            <p>
-. In a 14mL round-bottomed culture tube add 1.8mL selective dropout media and 0.2mL 20% glucose. <br>
-. Take 20-50uL from a previous overnight or passage culture and add it to the culture media.<br>
-. Incubate and shake at 37C at 250rpm for 2 days. <br>
-				Note: You can also do 3mL cultures (2.7mL S.D. media and 0.3mL 20% glucose) or larger cultures just make sure to dilute the glucose from 20% to 2%. <br>
+            For maximum effectiveness, final concentration should be approximately 8.5 M in PBS<br>
-				Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least 1 living cell is passaged.
-			</p>
+            Add the following to a 500 mL beaker and mix:
+            </p>
+            <p>
+                - 203 g guanidinium hydrogen chloride <br>
+                - 250 mL PBS solution* <br>
+                - Add dilute HCl to pH 7.4
+            </p>
+            <p>
+                *It is not necessary to filter or autoclave. <br>
+            *Alternatively add slightly less than 250 mL of PBS in order to buffer the solution to the appropriate volume, then add more dH2O as necessary.
+</p>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+<html><a name="Basic Cloning"></a>
+</html>
+    <h2> Basic Cloning </h2>
+        <h3> Polymerase Chain Reaction </h3>
+            <p>
+                All PCRs were done using a standard 50 μL reaction volume with GoTaq® Green Master Mix 2X purchased from PROMEGA Corporation. <br>
+                Mix the following in a 0.2 mL microcentrifuge tube on ice: <br>
+μL GoTaq® Green Master Mix 2X <br>
+-5 μL of 10 μM forward primer <br>
+-5 μL of 10 μM reverse primer <br>
+                    <250 ng of DNA template <br>
+                    QS 50 μl nuclease-free H2O <br>
+                Conduct the reaction in a thermocycler, adjusting anneal temperature and extension times accordingly. See your polymerase supplier protocol for more details on thermocycling.
+            </p>
+        <h3> Error-prone Polymerase Chain Reaction </h3>
+            <p>
+                Prepare 50 μL reaction: <br>
+μL 10X Mutazyme II Rxn Buffer <br>
+μL 40 mM dNTP mix (200 μM each final) <br>
+μL 20 μM forward primer <br>
+μL 20 μM reverse primer <br>
-		<h3> Glycerol Stocks </h3>
-			<p>
+μL Mutazyme II DNA polymerase (2.5 U/μL) <br>
-. Take 1-2mL from an overnight culture and transfer into a 1.5mL centrifuge tube. <br>
-. Spin down the culture at 3000rpm for 3 minutes. <br>
-. Decant the supernatant. <br>
-. Resuspend the cells in 500uL of 40% Glycerol and 500uL of Selective Dropout media or water. <br>
-. Transfer the resuspension to a cryogenic vial. <br>
-. Store the glycerol stock at -80C. <br>
-			</p>
-	<h2> Flow Cytometry </h2>
+.01 ng template <br>
-		<h3> Dilutions </h3>
+                QS 50 μL diH2O <br>
-			<p>
-. From an overnight culture measure the optical density. <br>
-. Take enough culture to make a 1mL aliquot with 0.4 optical density. <br>
-. Spin down the aliquot in a 1.5mL centrifuge tube at 3000rpm for 3 minutes. <br>
-. Decant the supernatant. <br>
-. Resuspend the cell pellet in 800uL of the appropriate selctive dropout media and 200uL of 20% Glucose. <br>
-. Transfer the new culture to a 14mL round-bottomed tube. <br>
-. Incubate and shake at 30C and 250rpm for at least 6 hours (1.2-1.6 optical density).
-			</p>
+                <br>
-		<h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3>
-			<p>
+                Program thermocycler as follows: <br>
-. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br>
+°C, 2 min <br>
-. Make an aliquot of 500uL of the dilution culture in a 1.5mL centrifuge tube. <br>
-. Spin down the aliquot at 3000rpm for 3 minutes. <br>
+°C, 30 sec <br>
-. Decant the supernatant. <br>
-. Resuspend the cell pellet in 500uL of PBS(F). <br>
-. Spin down the resuspension at 3000rpm for 3 minutes. <br>
-. Decant the supernatant. <br>
-. Resuspend the cell pellet in another 500uL of PBS(F).* <br>
-. Prepare the C6 Flow Cytometer by running a backflush cycle and a dH2O cycle. <br>
-. Load the sample onto the sip. <br>
-. Run the sample without 100,000 cell count. <br>
-. Repeat for all samples and make sure to change data cells otherwise the old data is earased. <br>
-. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a dH2O cycle. <br>
-				*For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.
-			</p>
+                XX °C*, 30 sec <br>
+°C, X min** <br>
+cycles <br>
+°C, 10 min <br>
+°C, hold <br>
+                <br>
+                *Adjust annealing temperature according to Tm of primer. <br>
+                **Adjust extension time according to the length of amplified DNA. <br>
+                <br>
+                Note: Use 0.01 ng of template (calculate by insert and not by total plasmid). <br>
+                Calculate amount of template to use as follows: <br>
+                (bp for amplified region) / (bp in total plasmid) = % amplified region <br>
+                (conc. of total plasmid) x (% amplified region as a decimal) = conc. of amplified region <br>
+                Note: Never pipette less than 0.5 μL. <br>
+                (0.01 ng of template) / (conc. of amplified region) = vol of template to add to PCR  <br>
+            </p>
+        <h3> Restriction Endonuclease Reaction (Digestion) </h3>
+            <p>
+                All restriction enzyme reactions were done using a 50 μl reaction volume. Restriction enzymes and buffers were purchased from New England Biolabs® Inc. <br>
+                Mix the following in a 0.2 mL PCR tube: <br>
+μg of DNA <br>
+μL of the appropriate 10X New England Biolab® Buffer <br>
+μL of each restriction enzyme (add last) <br>
+                    QS 50μL nuclease-free H2O <br>
+                Incubate the reaction for 1 hr <br>
+                Heat inactive the reaction at the appropriate temperature <br>
+<br>
+                Note: Thaw the restriction enzyme(s) on ice to improve shelf life. <br>
+            </p>
+        <h3> Ligation </h3>
+            <p>
+                T4 DNA Ligase and Buffer were purchased from New England Biolabs® Inc. <br>
+. Prepare the following in a 0.2 mL microcentrifuge tube: <br>
+.0 ng vector DNA* <br>
+.5 ng vector DNA* <br>
+μL 10X T4 DNA Ligase Buffer <br>
+μL T4 DNA Ligase <br>
+                    QS 20 μL diH2O <br>
+. Incubate the reaction at room temperature for 10-30 minutes or at 16 °C overnight. <br>
+. Heat inactivate at 65 °C for 10 minutes. <br>
+. Chill on ice before starting a transformation reaction. <br>
+<br>
+                *The exact amount of DNA is dependent on the number of base pairs. In order to conduct a proper reaction consult the New England Biolab Ligation Calculator at:
+                    http://nebiocalculator.neb.com/#!/
+            </p>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+<html><a name="Escherichia coli Protocols"></a>
+</html>
+    <h2> <i> Escherichia coli </i> Protocols (XL1-Blue and XL10-Gold) </h2>
+        <h3> Chemically Competent Cell Cultures </h3>
+            <p>
+Competent cells take two days to culture and aliquot. <br>
+           Day 1: <br>
+. Streak an aliquot of competent cells onto two LB-plates without antibiotics.* <br>
+. Incubate at 37 °C overnight. <br>
+                Day 2: <br>
+. In two 250 mL baffle flasks add 50 mL of SOB media. <br>
+. Scrape as many single colonies into either flask. <br>
+. Incubate and shake at 37 °C and 250 rpm for 2-3 hours. <br>
+. Check the optical density of the cells at 600 nm after 2 hours. <br>
+. Stop incubation when cultures reach approximately 0.5 optical density. <br>
+. Add the contents of the flask into separate 50 mL flat bottomed centrifuge tubes. <br>
+. Spin down the cells at 2500 rpm at 4 °C for 15 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cells in 16 mL of CCMB by pipetting or gently vortexing. <br>
+. Incubate the cells on ice for 20 minutes. <br>
+. Spin down the cells at 2500 rpm at 4 °C for 10 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cells in 4 mL of CCMB. <br>
+. Quickly aliquot the cells into 1.7 mL cryogenic vials or 1.5 mL centrifuge tubes.** <br>
+. Store the competent cell aliquots at -80 °C. <br>
+<br>
+                    *Streak in such a way that there should be individual colony growth and no clumps after the incubation. <br>
+                    **We did this in a -20 °C cold room and using an automated repeater pipette. The volume of each aliquot depends on the number of transformations you intend to do at a time. <br>
+<br>
+                    Note: After removing the cells from incubation keep them on ice or as cold as possible. <br>
+            </p>
+        <h3> Chemically Competent Cell Transformations </h3>
+            <p>
+.   Thaw competent <i> E. coli </i> cells on ice (XL1-Blue or XL10-Gold).* <br>
+.   Add 50 μL of competent cells to sterile 14 mL culture tube. <br>
+.   Add 1 μL (~100-200 ng)* of the mini-prep to each culture tube. <br>
+.   Equilibrate the cells on ice for 10 minutes. <br>
+.   Heat shock the cells at 42 °C for 30-45 seconds.** <br>
+.   Immediately place the cells back on ice for 3 minutes. <br>
+.   Add 250 μL LB media without antibiotics and shake at 250 rpm and 37 °C for 30 minutes. <br>
+.   Spread 10 μL and 290 μL on an appropriate LB-antibiotic plate. <br>
+.   Invert the plate and incubate at 37 °C overnight. <br>
+<br>
+                *The exact amount of DNA to add depends on your cell's transformation efficiency. However, it is acceptable to add a larger amount to increase the number of transformed cells. <br>
+                **Do not heat shock for an extended duration as this may damage and/or kill your cells.
+            </p>
+        <h3> Overnights </h3>
+            <p>
+. In a 14 mL round-bottom tube, add 3 mL of LB and 3 μL of 1000X antibiotic(s). <br>
+. Pick one isolated colony, do not collect satellites or colony clumps, with a pipette tip. <br>
+. Swirl the colony tip in the tube, there should be no visible cell clumps. <br>
+. Incubate and shake the tube at 37 °C at 250 rpm for 12-16 hours and no longer than 20 hours. <br>
+            </p>
+        <h3> DNA Extraction and Mini-Preps </h3>
+            <p>
+                All DNA Mini-Preps were prepared using EPOCH Mini-Prep Kits and following the supplied protocols.
+            </p>
+        <h3> Glycerol Stocks </h3>
+            <p>
+. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br>
+. Spin down the culture at 3000 rpm for 3 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of LB (no antibiotics) or water. <br>
+. Transfer the resuspension to a cryogenic vial. <br>
+. Store the glycerol stock at -80 °C. <br>
+            </p>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+<html><a name="Saccharomyces cerevisiae"></a>
+</html>
+    <h2> <i> Saccharomyces cerevisiae </i> (PyE1 Yeast) </h2>
+        <h3> Chemically Competent Cell Cultures </h3>
+            <p>
+                This process take four days in lab with a one day wait for incubation. <br>
+                 Day 1: <br>
+. Streak yeast cells onto a YPD plate.* <br>
+. Invert the plate and incubate at 30 °C for 2 days. <br>
+                Day 3: <br>
+. Add 50 mL of YPD liquid media into a 250 mL baffle flask. <br>
+. Swipe as many individual colonies as you can see into the YPD media.** <br>
+. Incubate and shake the culture at 30 °C at 250 rpm overnight approximately 24 hours. <br>
+                Day 4: <br>
+. Take an optical density measurement. <br>
+. In three 250 mL baffle flask add the portions of the overnight liquid culture. <br>
+. Dilute each culture to approximately 0.4 optical density with YPD. <br>
+. Incubate and shake the cultures at 30 °C at 250 rpm until the optical density reaches 1.2-1.6. <br>
+. Collect each culture into separate 50 mL flat-bottomed centrifuge tubes. <br>
+. Spin down the cells at 4000 x g for 5 minutes at 4 °C. <br>
+. Decant the supernatant. <br>
+. Resuspend the cells in 100 mL total for all three culture of dH2O. <br>
+. Combine the suspensions into two 50 mL flat-bottomed centrifuge tubes. <br>
+. Spin down the cells as above. <br>
+. Decant the supernatant. <br>
+. Resuspend each in 3 mL of 100 mM lithium acetate. <br>
+. Transfer both cultures into a single 15 mL conical centrifuge tube. <br>
+. Spin down the cells at 3000 rpm for 5 minutes. <br>
+. Resuspend the cells in 0.75 mL of 100 mM lithium acetate, total volume is roughly 2 mL. <br>
+. Qualitatively bring up the volume to 3.5 mL by adding 40% glycerol. <br>
+. Aliquot the cells into 1.5 mL centrifuge tubes or 1.7 mL cryogenic vials.*** <br>
+<br>
+                *Streak in such a way that there are individual colonies visible on the plate without clumps or satellite colonies. <br>
+                **Collect only individual visible colonies. Do not collect clumps or satellite colonies. <br>
+                ***The volume of aliquots depends on the number to transformations you intend to do at a time. <br>
+            </p>
+        <h3> Chemically Competent Transformations </h3>
+            <p>
+                 This protocol assumes a 50 μL aliquot of yeast competent cells were made. Furthermore, this protocol prepares enough cells for six yeast transformations. <br>
+                <br>
+. Add the following to 50 μL of yeast competent cells: <br>
+μL of polyethylene glycol - 3350 (PEG-3350) <br>
+μL of 1 M lithium acetate <br>
+μL of milliQ H2O <br>
+. Mix the mixture by gently pipetting or vortexing. <br>
+. Aliquot 59 μL of the mixture into a 0.2 mL microcentrifuge tube. <br>
+. Add 1 μL (~100-200 ng) of DNA.* <br>
+. Mix the mixture by gentle pipetting or vortexing. <br>
+. Incubate the mixture at 30 °C for 30 minutes. <br>
+. Heat shock the mixture at 42 °C for 20 minutes. <br>
+. Spin down the cells in a microcentrifuge for ~1 minute. <br>
+. Decant the supernatant. <br>
+. Resuspend the cell pellets in 200 μL of dH2O. <br>
+. Spin down the cells in a microcentrifuge for ~1 minute. <br>
+. Resuspend the cell pellets in 200 μL of dH2O. <br>
+. Plate 50-150 μL of the mixture onto an appropriate selective dropout media plate. <br>
+. Invert and incubate at 30 °C for 2 days. <br>
+<br>
+                *The exact amount of DNA depends on the transformation efficiency of your competent cells.
+            </p>
+        <h3> Overnight Culturing </h3>
+            <p>
+. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br>
+. Swipe 3 isolated yeast colonies and add them to the culture tube media. <br>
+. Incubate and shake at 37 °C at 250 rpm for 2 days. <br>
+<br>
+                Note: You can also make 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%.
+            </p>
+        <h3> Culture Passaging </h3>
+            <p>
+. In a 14 mL round-bottomed culture tube add 1.8 mL selective dropout media and 0.2 mL 20% glucose. <br>
+. Take 20-50 μL from a previous overnight or passage culture and add it to the culture media.<br>
+. Incubate and shake at 37 °C at 250 rpm for 2 days. <br>
+<br>
+                Note: You can also do 3 mL cultures (2.7 mL S.D. media and 0.3 mL 20% glucose) or larger cultures, just make sure to dilute the glucose from 20% to 2%. <br>
+                Note: The exact amount of culture that you take from a previous culture is irrelevant as long as at least one living cell is passaged.
+            </p>
+        <h3> Glycerol Stocks </h3>
+            <p>
+. Take 1-2 mL from an overnight culture and transfer into a 1.5 mL centrifuge tube. <br>
+. Spin down the culture at 3000 rpm for 3 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cells in 500 μL of 40% glycerol and 500 μL of selective dropout media or water. <br>
+. Transfer the resuspension to a cryogenic vial. <br>
+. Store the glycerol stock at -80 °C. <br>
+            </p>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+<html><a name="Flow Cytometry"></a>
+</html>
+    <h2> Flow Cytometry </h2>
+        <h3> Dilutions </h3>
+            <p>
+. From an overnight culture measure the optical density at 660 nm by making 1:10 dilutions. <br>
+. Take enough culture to make a 1 mL aliquot with an OD of 0.4. <br>
+. Spin down the aliquot in a 1.5 mL centrifuge tube at 3000 rpm for 3 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cell pellet in 800 μL of the appropriate selective dropout media and 200 μL of 20% glucose. <br>
+. Transfer the new culture to a 14 mL culture tube. <br>
+. Incubate and shake at 30 °C and 250 rpm for at least 6 hours (1.2-1.6 optical density).
+            </p>
+        <h3> Preparations for Analysis using C6 Accuri Flow Cytometer </h3>
+            <p>
+. From the dilution previously made, measure the optical density, roughly 1.2-1.6. <br>
+. Make an aliquot of 500 μL of the dilution culture in a 1.5 mL centrifuge tube. <br>
+. Spin down the aliquot at 3000 rpm for 3 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cell pellet in 500 μL of PBSF. <br>
+. Spin down the resuspension at 3000 rpm for 3 minutes. <br>
+. Decant the supernatant. <br>
+. Resuspend the cell pellet in another 500 μL of PBSF.* <br>
+. Prepare the C6 Accuri Flow Cytometer by running a backflush cycle and a diH2O cycle. <br>
+. Load the sample onto the sip. <br>
+. Run the sample with 100,000 cell count. <br>
+. Repeat for all samples and make sure to change data cells otherwise the old data will be erased. <br>
+. Once finished, run a cleaning cycle with Accuri approved cleaning solution, then run a diH2O cycle. <br>
+<br>
+                *For special cases do not resuspend all samples, instead resuspend immediately before running the sample through the flow cytometer.
+            </p>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+<html><a name="Fluorescence-Activated Cell Sorting"></a>
+</html>
-	<h2> Fluorescence Activated Cell Sorting </h2>
+    <h2> Fluorescence-Activated Cell Sorting </h2>
-		<h3> Final Preparations </h3>
+      <h3>Dilution of Cells:</h3>
 			<p>
-				Sample Prep:
+. Take OD600 of the cultures.<br>
+. Calculate volume to spin down for OD of 0.4 in appropriate volume (typically 1 mL) - C1V1=C2V2 <br>
+. Spin down appropriate volume in eppendorf tubes for 3 min at 3000 rpm.<br>
+. Aspirate off supernatant.<br>
+. Resuspend pellet in 1 mL C-Ura + 2% glucose (or other appropriate media).<br>
+. Shake in 14 mL culture tube at 30 °C for 6 hrs. <br>
+</p>
-					Spin down samples and negative control (5000 RPM, 1 min), keeping in mind the library size. Aspirate off supernatant. Resuspend in PBSF. Spin down cells. Aspirate off supernatant. Resuspend in PBSF. <br>
+		<h3>Sample Prep:</h3>
-						<br>
-. Open the “iGEM Template” file in the FACS Software and change name to current date and sort cycle<br>
+			<p>
-. Make sure the stream is stable (look for green light in bottom right corner). If not, run the Sort Calibration order. <br>
+. Transfer 500 μL of samples and negative control to eppendorf tubes.<br>
-. Run Negative Control from the PyE1 cells. <br>
+. Spin down cells (3000 rpm, 3 min). <br>
-    						 a. Load cells onto carrier and into the machine. Press play button on screen. <br>
+. Aspirate off supernatant. Resuspend in PBSF. <br>
-    						 b. Set gate around lower left quadrant of cells to ensure single cell analysis using Forward Scatter Area and Side Scatter Area as your axes. Make sure oval gate covers around 80% of cell population. <br>
+. Spin down cells (3000 rpm, 3 min).<br>
-     						 c. Set second gate on the first gated population by double-clicking on the gated population and using Forward Scatter Height and Forward Scatter Width as your axes. You will notice two distinct populations. Try to focus on the single cell portion of the plot. <br>
+. Aspirate off supernatant. Resuspend in PBSF.<br>
-							NOTE: If you see a large portion of the second gated population existing near the upper right edge of the first gate, you may need to enlarge the first gate to fit more of the population. <br>
-     						 d. Press record. Record 100,000 events and stop run. Move to Next Tube. <br>
-. Run first control (Gene clone) <br>
-     						 a. Follow step 3 to run the first control <br>
-. Run first library sample <br>
-    						a. Follow steps 3b and 3c to set first two gates correctly. <br>
-     						b. Set final gate for sort which includes top 1% of GFP producers from second gated population. <br>
-     						c. Use final gate to set up the sort. <br>
-     						d. Select sort conditions at the bottom of the screen. <br>
-    						e. Insert and load collection tube. <br>
-     						f. Record 100,000 events, and sort 10x the library size <br>
-. Run Bleach and diH2O through FACS to avoid cross-contamination. <br>
 			</p>
+		<h3>Using the Fluorescence-Activated Cell Sorter</h3>
+			<p>
+. Load the “iGEM Template” file in the FACS Software.<br>
+. Make sure the stream is stable.<br>
+. Run all controls and record 100,000 events for analysis. <br>
+. While running controls, set FSC/SSC and FSC-H/FSC-W gates.<br>
+. Run library and record 100,000 events for analysis.<br>
+. Set gate for top 1.00% of GFP fluorescence.<br>
+. Sort cells falling in all three gates. Sort ten-fold over library size.<br>
+. Run bleach and diH2O through FACS to avoid cross-contamination.<br>
-	<h2> Protein Expression </h2>
+    			</p>
+<html><a name="Protein Expression"></a>
+</html>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+    <h2> Protein Expression </h2>
+        <h3> Overnight Cultures </h3>
-		<h3> Overnight Cultures </h3>
 <p>
-. Add 25mL TB and 25uL Kan to a 250mL baffled flask <br>
-. Stab a glycerol stock with a p1000 pipette and swirl in the flask of media <br>
+. Add 25 mL TB and 25 μL 1000X Kan to a 250 mL baffled flask. <br>
-. Put flask in 37C shaker overnight
+. Stab a glycerol stock with a P1000 pipette and swirl in the flask of media. <br>
+. Put flask in 37 °C shaker at 250 rpm for 16-20 hrs.<br>
 </p>
                  <h3> Protein Expression </h3>
 <p>
-. Add 500uL 1000x Kanamycin to 500mL TB in 2L baffled flask <br>
-. Transfer 10mL overnight culture to TB <br>
+. Add 500 μL 1000X Kanamycin and 1 mL MgSO4 to 500 mL TB in 2 L baffled flask. <br>
-. Shake at 37C (DO WE NEED THE RPM?) <br>
+. Transfer 10 mL overnight culture to TB. <br>
-. Remove flask from shaker when optical density is between 0.5 and 0.8 <br>
+. Shake at 37 °C and 250 rpm until OD600 is between 0.5 and 0.8. <br>
-. Allow flask to rest at room temp for 30 min <br>
+. Allow flask to rest at room temp for 30 min. <br>
-. Add 125uL 1M IPTG <br>
+. Add 125 μL 1 M IPTG. <br>
-. Shake flask at 18C overnight
+. Shake flask at 18 °C for ~16-20 hrs.
 </p>
-		<h3> Protein Extraction and Purification </h3>
+        <h3> Protein Extraction and Purification </h3>
 <p>
-. Transfer cell culture to centrifuge flask <br>
+. Transfer cell culture to centrifuge tube. <br>
-. Centrifuge culture at 4000g for 10 min <br>
+. Centrifuge culture at 4000 x g for 10 min. <br>
-. Discard supernatant <br>
+. Discard supernatant. <br>
-. Resuspend pellet in 25mL lysis buffer <br>
+. Resuspend pellet in 25 mL wash buffer and add 250 μL of 100X PMSF, 250 μL of 100 mg/mL lysozyme, and 250 μL of 10 mg/mL DNAse. <br>
-. Add 250uL of 100x PMSF, 250uL of 100mg/mL lysozyme, and 250 uL of 10mg/mL DNAse <br>
+. Sonicate sample with 0.25 inch probe for 5 min at 70% amplitude with 20 sec on and off pulses. <br>
-. Sonicate sample with 0.25inch probe for 5 min at 70% amplitude with 20 sec on and off pulses <br>
+. Take 50 μL total sample. <br>
-. Take 50uL total sample <br>
+. Transfer lysate to SS-34 centrifuge tube. <br>
-. Transfer lysate to SS-34 centrifuge tube <br>
+. Centrifuge for 30 min at 18000 x g. <br>
-. Centrifuge for 30 min at 18000g <br>
+. Take 50 μL soluble sample.
-. Take 50uL soluble sample
 </p>
-		<h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3>
+        <h3> Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography) </h3>
 <p>
-. Add 5mL 50%(v/v) nickel resin in ethanol to 25mL column and allow to settle to ~2.5mL(CV) <br>
-. Rinse with 10CV H20 <br>
+. Add 5 mL 50%(v/v) nickel resin in ethanol to a 25 mL gravity flow column and allow to settle to 2.5 mL(CV). <br>
-. Equilibrate with 10CV lysis buffer <br>
+. Rinse with 10CV dH2O. <br>
-. Load sample onto column <br>
+. Equilibrate with 10CV lysis buffer. <br>
-. Wash column with 15CV lysis buffer <br>
+. Load sample onto column. <br>
-. Perform 2 additional wash steps with 15CV <br>
+. Wash column with 15CV lysis buffer. <br>
-. Elute sample in 10CV elution buffer and collect eluate <br>
+. Perform 2 additional wash steps with 15CV. <br>
-. Take 50uL pure sample
+. Elute sample in 10CV elution buffer and collect eluate. <br>
+. Take 50 μL pure sample.
 </p>
-		<h3> Size Exclusion Chromatography (S.E.C.) </h3>
-	<h2> Stability Analysis </h2>
+        <h3> Size Exclusion Chromatography (SEC) </h3>
+<p>
+. Concentrate sample to as high as possible without inducing protein aggregation.<br>
+. Pre-equilibrate Superdex 75 column with 48 mL PBS. <br>
+. Inject 500 μL sample onto column. <br>
+. Run 36 mL PBS through column at 0.5 mL/min, collecting 1 mL fractions. <br>
+. Verify presence of protein in fractions by measuring concentration on Nanodrop and running SDS-PAGE (15 kDa protein should elute at ~13 mL).<br>
+. Pool fractions containing protein.
-		<h3> Thermal Melts </h3>
+</p>
-		<h3> Guanidinium Hydrogen Chloride Melts</h3>
+<html>
+<br><a href="#top">Back To Top</a> <br>
+</html>
+<html><a name="Stability Analysis"></a>
+</html>
+    <h2> Stability Analysis </h2>
+        <h3> Circular Dichroism: Wavelength Scan </h3>
+<p>
+. Load 1mm cuvette with 400 μL protein solution onto CD. <br>
+. Take wavelength scan: <br>
+nm-190 nm <br>
+    sample every 1 nm <br>
+    averaging time 3 sec <br>
+scan <br>
+    step scan <br>
+°C <br>
+. Record wavelength which gives strongest signal (222 nm).
+</p>
+	<h3> Circular Dichroism: Guanidine Melt </h3>
+<p>
+. Load 1 cm cuvette containing 1.996 mL of 0.05 mg/mL protein solution and stirrer onto CD. <br>
+. Prepare 8 mL of 0.05 mg/mL protein in concentrated guanidine solution. <br>
+. Set up Automixer with guanidine solution on one syringe and waste tube on other syringe. <br>
+. Titrate up to 6 M guanidine, taking a CD measurement at 222 nm every 0.15 M interval. <br>
+. Also measure the fluorescence at 280 nm to ensure the total protein concentration is not changing.
+</p>
+<html>
+<a href="#top">Back To Top</a>
+</html>

Team:Washington/Protocols

From 2014.igem.org

Latest revision as of 03:40, 18 October 2014

Protocols

Contents

Media, Plates, and Solutions

Competent Cell Media Buffer (CCMB)

Super Optimal Broth (SOB)

Phosphate Buffered Saline (PBS) Solution

PBSF (PBS for Flow)

Yeast Extract Peptone Dextrose (YPD)

Selective Dropout Media, C-Uracil and C-Histidine (C-Ura and C-His)

Guanidinium Hydrogen Chloride

Basic Cloning

Polymerase Chain Reaction

Error-prone Polymerase Chain Reaction

Restriction Endonuclease Reaction (Digestion)

Ligation

Escherichia coli Protocols (XL1-Blue and XL10-Gold)

Chemically Competent Cell Cultures

Chemically Competent Cell Transformations

Overnights

DNA Extraction and Mini-Preps

Glycerol Stocks

Saccharomyces cerevisiae (PyE1 Yeast)

Chemically Competent Cell Cultures

Chemically Competent Transformations

Overnight Culturing

Culture Passaging

Glycerol Stocks

Flow Cytometry

Dilutions

Preparations for Analysis using C6 Accuri Flow Cytometer

Fluorescence-Activated Cell Sorting

Dilution of Cells:

Sample Prep:

Using the Fluorescence-Activated Cell Sorter

Protein Expression

Overnight Cultures

Protein Expression

Protein Extraction and Purification

Nickel Nitrotriacetic Acid Chromatography (Nickel-NTA Chromatography)

Size Exclusion Chromatography (SEC)

Stability Analysis

Circular Dichroism: Wavelength Scan

Circular Dichroism: Guanidine Melt