Team:Washington/Protocols

From 2014.igem.org

(Difference between revisions)
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<h3> CCMB </h3>
<h3> CCMB </h3>
-
<h3> LB-Agar </h3>
+
<p>
-
<p align="left">
+
        100g Glycerol (liquid) <br>
-
10 g tryptone
+
          10mL x 1M PotassiumAcetate <br>
-
5 g yeast extract
+
          11.8g CaCl2*H2O <br>
-
10 g NaCl
+
          4g MnCl2 <br>
-
15 g agarose
+
          2g MgCl2 <br>
-
1 Liter diH2O
+
    Mix in 1L diH2O <br>
-
Autoclave in two 500 ml bottles (liquid cycle, 20 min)
+
    Sterile Filter or Autoclave (20min at 121C at 20psi) in a 1L bottle
-
</p>
+
</p>
<h3> Luria Broth </h3>
<h3> Luria Broth </h3>
-
<p align="left">
+
 
-
6.25 g LB mix
+
<p>
-
250 ml diH2O
+
10 g tryptone <br>
-
Autoclave in 500 ml bottle (liquid cycle, 20 min)
+
5 g yeast extract <br>
-
</p>
+
10 g NaCl <br>
 +
1000 ml diH2O <br>
 +
Autoclave in two 500 ml bottle (20 min at 121C at 20psi)
 +
</p>
 +
 
 +
<h3> LB-Agar </h3>
 +
 
 +
<p>
 +
1000ml L.B. as above <br>
 +
15 g agar <br>
 +
1 Liter diH2O <br>
 +
Autoclave in two 500 ml bottles (20 min at 121C at 20psi)
 +
</p>
 +
 
 +
 
 +
 
<h3> Super Optimal Broth (S.O.B.) </h3>
<h3> Super Optimal Broth (S.O.B.) </h3>
 +
                       
 +
                        <p>
 +
        20g BactoTryptone <br>
 +
        5g BactoYeast Extract <br>
 +
        10mL x 1M NaCl <br>
 +
          2.5mL x 1M KCl <br>
 +
    Mix in 1L of diH2O <br>
 +
    Sterile filter or Autoclave (20 min at 121C at 20psi) in a 1L bottle
 +
                        </p>
<h3> Guanidinium Hydrogen Chloride </h3>
<h3> Guanidinium Hydrogen Chloride </h3>
 +
 +
<p>
 +
For maximum effectiveness, final concentration should be 8.5M in a Phosphate Buffered Saline Solution <br>
 +
203g Guanidinium Hyrdogen Chlordie <br>
 +
250mL PBS solution <br>
 +
</p>
 +
 +
<h3> PBS(F) </h3>
 +
 +
<p>
 +
8g NaCl <br>
 +
1.44g Na_2HPO_4 <br>
 +
0.8g KCl <br>
 +
0.24g KH_2PO_4 <br>
 +
Mix in 1L of diH2O and buffer to pH 7.4 <br>
 +
Sterile Filter or Autoclave (20 min at 121C at 20psi) in a 1L bottle
 +
</p>
<h3> YPD </h3>
<h3> YPD </h3>
-
<h3> Yeast Liquid Cultures </h3>
+
<p>
 +
 +
</p>
-
<h3> PBSF </h3>
+
<h3> C-Uracil and C-Histidine </h3>
 +
 
 +
<p>
 +
Synethesized by the Yeast Resource Center at the Univeristy of Washington's
 +
Department of Genome Sciences and Department of Biochemistry.
 +
</p>
<h2> Basic Cloning </h2>
<h2> Basic Cloning </h2>
<h3> Polymerase Chain Reactions </h3>
<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: <br>
 +
25uL GoTaq® Green Master Mix 2X <br>
 +
1-5uL of 10uM Forward primer <br>
 +
1-5uL of 10uM Reverse primer <br>
 +
<250ng of DNA template <br>
 +
Nuclease-Free Water to 50μl <br>
 +
In a thermocyler conduct the reaction...
 +
</p>
 +
<h3> Restriction Endonuclease Reaction (Digestion) </h3>
<h3> Restriction Endonuclease Reaction (Digestion) </h3>
-
+
 +
<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>
 +
1uL of each Restriction Enzyme, add the RE last <br>
 +
1ug of DNA <br>
 +
5uL 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>
 +
Thaw the restriction enzyme(s) on ice to improve shelf life <br>
 +
 +
</p>
 +
 
<h3> Ligation </h3>
<h3> Ligation </h3>
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<h3> Competent Cell Culturing </h3>
<h3> Competent Cell Culturing </h3>
 +
 +
<p>
 +
Competent Cells take two days to culture and aliquot.
 +
Day 1:
 +
1. Streak an aliquot of Compentent Cells onto two LB-plates without anti-biotics. <br>
 +
2. Incubate at 37C overnight. <br>
 +
Day 2: <br>
 +
1. In two 250mL baffle flask add 50mL of S.O.B. media. <br>
 +
2. Scrape as many single colonies into either flask. <br>
 +
3. Incubate and shake at 37C and 250rpm for 2-3 hours. <br>
 +
4. Check the optical density of the cells at 550nm after 2 hours. <br>
 +
5. Stop incubation when cultures reach approximately 0.5 optical density. <br>
 +
6. Add the contents of the flask into separate 50mL flat bottomed centrifuge tubes. <br>
 +
7. Spin down the cells at 2500rpm at 4C for 15 minutes. <br>
 +
8. Decant the supernatant. <br>
 +
9. Resuspend the cells in 16mL of CCMB by pipetting or gentle vortexing. <br>
 +
      10. Incubate the cells on ice for 20 minutes. <br>
 +
      11. Spin down the cells at 2500rpm at 4C for 10 minutes. <br>
 +
      12. Decant the supernatant. <br>
 +
      13. Resuspend the cells in 4mL of CCMB. <br>
 +
      14. Quickly aliquot the cells into 1.7mL cryovials or 1.5mL centrifuge tubes.* <br>
 +
      15. Store the competent cell aliquots at -80C. <br>
 +
 +
*We did this in a -20C cold room and using a automated repeater pipette. <br>
 +
**After removing the cells from incubation keep them on ice or as cold as possible. <br>
 +
</p>
 +
<h3> Competent Cell Transformations </h3>
<h3> Competent Cell Transformations </h3>
-
<p align="left">
+
 
-
1.  Thaw competent E.coli cells on ice (XL1-Blue for cloning) <br>
+
<p>
-
2.  Add 50 uL of competent cells to sterile 14 mL Falcon culture tubes <br>
+
1.  Thaw competent E.coli cells on ice (XL1-Blue for cloning) <br>
-
3.  Add 1 uL of the miniprep to each culture tube <br>
+
2.  Add 50 uL of competent cells to sterile 14 mL Falcon culture tubes <br>
-
4.  Equilibrate the cells on ice for 10 min <br>
+
3.  Add 1 uL of the miniprep to each culture tube <br>
-
5.  Heat shock the cells at 42C for 30-45 seconds <br>
+
4.  Equilibrate the cells on ice for 10 min <br>
-
6.  Immediately place the cells back on ice for 3 min <br>
+
5.  Heat shock the cells at 42C for 30-45 seconds <br>
-
7.  Add 250 uL LB media and shake at 250 rpm and 37C for 30 min <br>
+
6.  Immediately place the cells back on ice for 3 min <br>
-
8.  Plate 10 ul and 290 ul of the recovered cells onto LB-agar plates supplemented with appropriate antibiotics <br>
+
7.  Add 250 uL LB media and shake at 250 rpm and 37C for 30 min <br>
-
9.  Invert and incubate at 37C overnight
+
8.  Plate 10 ul and 290 uL <br>
-
</p>
+
9.  Invert and incubate at 37C overnight
 +
</p>
 +
 
<h3> Plating </h3>
<h3> Plating </h3>
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<h3> Thermal Melts </h3>
<h3> Thermal Melts </h3>
-
<h3> Guanidinium Hydrogen Chloride Melts</h3>
+
<h3> Guanidinium Hydrogen Chloride Melts</h3>
-
</html>
+

Revision as of 03:10, 12 October 2014

UW Homepage Official iGEM website

Contents

Protocols

Media, Plates and Solutions

CCMB

100g Glycerol (liquid)
10mL x 1M PotassiumAcetate
11.8g CaCl2*H2O
4g MnCl2
2g MgCl2
Mix in 1L diH2O
Sterile Filter or Autoclave (20min at 121C at 20psi) in a 1L bottle

Luria Broth

10 g tryptone
5 g yeast extract
10 g NaCl
1000 ml diH2O
Autoclave in two 500 ml bottle (20 min at 121C at 20psi)

LB-Agar

1000ml L.B. as above
15 g agar
1 Liter diH2O
Autoclave in two 500 ml bottles (20 min at 121C at 20psi)


Super Optimal Broth (S.O.B.)

20g BactoTryptone
5g BactoYeast Extract
10mL x 1M NaCl
2.5mL x 1M KCl
Mix in 1L of diH2O
Sterile filter or Autoclave (20 min at 121C at 20psi) in a 1L bottle

Guanidinium Hydrogen Chloride

For maximum effectiveness, final concentration should be 8.5M in a Phosphate Buffered Saline Solution
203g Guanidinium Hyrdogen Chlordie
250mL PBS solution

PBS(F)

8g NaCl
1.44g Na_2HPO_4
0.8g KCl
0.24g KH_2PO_4
Mix in 1L of diH2O and buffer to pH 7.4
Sterile Filter or Autoclave (20 min at 121C at 20psi) in a 1L bottle

YPD

C-Uracil and C-Histidine

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

Basic Cloning

Polymerase Chain Reactions

All PCRs were done using a standard 50uL reaction volume.
PCRs were done using GoTaq Green Master Mix 2X purchased from PROMEGA Corporation.
Protocols for the PROMEGA GoTaq Green Master Mix 2X:
Mix the following in a 0.2mL PCR tube:
25uL GoTaq® Green Master Mix 2X
1-5uL of 10uM Forward primer
1-5uL of 10uM Reverse primer
<250ng of DNA template
Nuclease-Free Water to 50μl
In a thermocyler conduct the reaction...


Restriction Endonuclease Reaction (Digestion)

All restriction enzyme reactions were done using a 50ul reaction volume.
Restriction enzymes and buffers were purchased from New England Biolabs Incorporated.
Protocols for various New England Biolab restriction enzyme reactions:
Mix the following in a 0.2mL PCR tube:
1uL of each Restriction Enzyme, add the RE last
1ug of DNA
5uL of the appropriate 10X New Englan Biolab Buffer
Nuclease-Free Water to 50uL
Incubate the reaction for 1hr
Heat inactive the the reaction at the appropriate temperature
Notes: Add the restriction enzyme(s) to the reaction last
Thaw the restriction enzyme(s) on ice to improve shelf life

Ligation

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

Competent Cell Culturing

Competent Cells take two days to culture and aliquot. Day 1: 1. Streak an aliquot of Compentent Cells onto two LB-plates without anti-biotics.
2. Incubate at 37C overnight.
Day 2:
1. In two 250mL baffle flask add 50mL of S.O.B. media.
2. Scrape as many single colonies into either flask.
3. Incubate and shake at 37C and 250rpm for 2-3 hours.
4. Check the optical density of the cells at 550nm after 2 hours.
5. Stop incubation when cultures reach approximately 0.5 optical density.
6. Add the contents of the flask into separate 50mL flat bottomed centrifuge tubes.
7. Spin down the cells at 2500rpm at 4C for 15 minutes.
8. Decant the supernatant.
9. Resuspend the cells in 16mL of CCMB by pipetting or gentle vortexing.
10. Incubate the cells on ice for 20 minutes.
11. Spin down the cells at 2500rpm at 4C for 10 minutes.
12. Decant the supernatant.
13. Resuspend the cells in 4mL of CCMB.
14. Quickly aliquot the cells into 1.7mL cryovials or 1.5mL centrifuge tubes.*
15. Store the competent cell aliquots at -80C.
*We did this in a -20C cold room and using a automated repeater pipette.
**After removing the cells from incubation keep them on ice or as cold as possible.


Competent Cell Transformations

1. Thaw competent E.coli cells on ice (XL1-Blue for cloning)
2. Add 50 uL of competent cells to sterile 14 mL Falcon culture tubes
3. Add 1 uL of the miniprep to each culture tube
4. Equilibrate the cells on ice for 10 min
5. Heat shock the cells at 42C for 30-45 seconds
6. Immediately place the cells back on ice for 3 min
7. Add 250 uL LB media and shake at 250 rpm and 37C for 30 min
8. Plate 10 ul and 290 uL
9. Invert and incubate at 37C overnight

Plating

Overnights

DNA-Extraction and mini-preps

Glycerol Stocks

Saccharomyces cerevisiae (PYE1 Yeast)

Transformations

OVernight Culturing and Passaging

Glycerol Stocks

Flow Cytometry and Fluorescence Activated Cell Sorting

Dilutions

Final Preparations

Protein Expression

Overnight Cultures

Protein Extraction and Purification

Nickel Nitrotriacetic Acid Chromatography

Size Exclusion Chromatography (S.E.C.)

Stability Analysis

Thermal Melts

Guanidinium Hydrogen Chloride Melts

Retrieved from "http://2014.igem.org/Team:Washington/Protocols"