Team:MIT/Protocols

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Protocols

Cloning	Tissue Culture	Miscellaneous Lab	Non-Lab
GELS GOLDEN GATE LR GATEWAY MAKNG LIQUID CULTURE MIDI PREP MINI Prep PCR RESUSPENDING PCR PRIMERS RESTRICTION DIGEST TRANSFORMATION BP REACTION ANNEALING AND KINASING OLIGOS MAKING CELL STOCKS MAKING SOC POURING GG DONOR PLATES PREPARING LB AGAR PLATES			MIT iGEM cookie preparation

Example

Example

Example

MAKING LIQUID CULTURE

MINI-PREP:
Prepare culture in a 15mL, round bottom tube.
Add 3-5mL LB
Add 3-5uL (respectively) of antibiotic (Ampicillin, Kanamycin, etc.)
Pick colony using a pipette tip. Eject tip into tube (tip should remain in tube).

MIDI-PREP:
Prepare culture in a 500mL Erlenmeyer flask.
Add 50mL LB
Add 50uL of antibiotic (Ampicillin, Kanamycin, etc.)
Pick colony using a pipette tip. Eject tip into tube (tip should remain in tube).

LR GATEWAY

Pro Tips
Run at the larger scale
ALWAYS kill it with PRoteinase K
Transform 4 uL of the reaction
Use ALL the transformation tricks
Plate ALL of it
ALWAYS run a PUC19 control for transformations

Instructions for newbies:
1.5 uL of 10 fM Dest
1.5 uL of 5 fM promoter
1.5 uL of 5 fM gene
1.5 uL of H2O
1.5 uL of LR clonase
Pipette up and down. Incubate at room temperature overnight.
Next day: Add 1.5 uL of proteinase K. Incubate for 15 minutes at 37C, then transform.

Instructions:
USE 3x VOLUME OF EVERYTHING (at least for now)
Use nanodrop to measure concentration of pEntry vectors, make 5 femtomolar working solution of each pEntry.
Excel sheet set up to calculate the necessary volumes
LR (concentration) calculations.xlsx
Sample calculation:Combine into 1 aliquot:
1uL of 5 fmol of pENTR_L4_Promoter_R1
1uL of 5 fmol pENTR_L1_Gene_L2
1uL of 10 fmol pDEST_R4_R2
3uL Total

WARNING: KEEP ALIQUOTED LR CLONASE MIX AT -80 AT ALL TIMES!
Add 0.5uL LR Clonase Enzyme
Additionally, remember to mix your reactions well after all elements have been added (use a 10 uL pipette set to 3 or 4 uL, then just pipette up and down gently)
Leave at room temperature for minimum of 16 hours, maximum of 24 hours.
transform just 1 uL of the reaction (This should result in around 50-500 colonies (more often than not closer to 500) with high (~90-95%) efficiency)
or storage -20C freezer.

GOLDEN GATE

50 ng of each piece of DNA being joined
Use nanodrop to find concentration in ng/ul, then divide 50 by that concentration to find the required volume of DNA:
Conc: x ng/uL
Vol: 50/x uL
NOTE: If GGDonr is too concentrated, dilute it with EB or water.
NOTE: Ligase buffer does not like to be freeze-thawed, so use one-time-use aliquots.

x1 uL of DNA1
x2 uL of DNA2

y uL (100ng) Donor
2ul 10X T4 Ligase Buffer
2ul 10X BSA
1ul BsaI (enzyme) HC (high concentration)
1ul T4 Ligase (enzyme) HC (high concentration)

fill to 20uL with SDIH20 (put water in before the buffer and enzymes)
-------------
20ul total
(NOTE: Make sure that Buffer and Enzyme added last, enzyme after buffer)

Take a p20, set it to 10uL and then pipet up and down.

THERMOCYCLER:
(Protocol EBGG)

37C for 5min
Part 1
50X:
37C for 2.5min
4C for 0.5min
16C for 5.5min
Part 2
37C for 10 min
80C for 20 min
4C hold (for 8+ hours)

(Check protocol by looking up the paper or other online GG protocols)

Gels

GELS
Preparing the Gel
Dissolve UltraPure agarose to a final concentration of 1%(by mass) in TAE buffer in a glass bottle.
Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously. ~1 minute/200ml solution
Place the solution in a warm water bath for 5 mins.
Add 10 µl SYBRSafe (1:10000) per 100 ml of the solution and mix well.
Pour 50ml of solution per small gel tray. (the gel trays and combs should be pre-cleaned with water and wiped dry).
-Note for combs: 15-well combs hold about 6 ul liquid, 12-well combs hold about 15 ul, 8-well combs hold about 20 ul
-Taping two 8-well comb wells together results in a well that holds up to 100 ul
-Taping three 8-well comb wells together result in a well that holds up to 200 ul
Use 120ml per large gel tray. [need to update amounts]
For the small set: small trays hold 20ml, large trays hold 50ml
Wait for the gels to solidify. ~15 mins
Label and store at 4C.

Running the Gel
When doing gel extraction, it is important to run both an analytical gel (to view under UV) and an extraction gel (from which bands are excised). UV damages DNA, and so we dont want to expose our extracted DNA.
Analytical Gel:
The analytical gel should have between 20 and 100 ng of DNA in each well. It should be an exact copy of the extraction gel with respect to position, voltage, and run time.
Extraction Gel:
This should be the rest of the digestion(s).
The analytical and extraction gels can technically be part of the same physical gel. Make sure to separate with a razor blade before imaging.
Refer to Gel Prep protocol above to determine the amounts of liquid to load for the specific well.
Appropriate Hyperladder to be used for PCR product which is linear. Usually Hyperladder I will be used.
While casting gel, add two sets of lanes; use one set to load an analytical gel.
Add 2ul gel loading buffer (Orange G 6X; it helps DNA sink into the bottom of the well) to DNA.
Make sure there is enough 1xTAE in the plate holder.
Load 5.0ul of appropriate hyperladder to one of the lanes.
Load appropriate amount of DNA - As much as possible! Usually 15-18ul - (mixed with the buffer) in each well.
Set the timer and voltage to 100V and 25 min.

Analytical Gel Annotation
The following things need to be added to the analytical gel image BEFORE it is posted to the wiki:
Label each lane with part number and amount of DNA loaded
Label each band with length and proposed identification
Include wt% agarose, run time, and voltage

Gel Extraction Protocol using Zymo kit (preferred if available)
Place the extraction gel on the blue light table.
Cut out the appropriate bands. Place into 2mL microtube(s). Try to cut out as small a piece as possible while still getting all the DNA.
Weigh gel slice (tare with empty microtube). Add 3 volumes of ADB buffer per mg of gel (so a 100mg gel gets 300 uL of ADB buffer).
Incubate at 55C for 10 minutes. Make sure that the gel is completely dissolved.
Add dissolved gel solution to Zymo column in collection tube. Max volume is 800 uL at a time.
Spin 14000 rpm for 30 sec.
Discard liquid in collection tube.
Repeat step 5-7 if had more than 800 uL dissolve gel.
Add 200 uL DNA wash buffer.
Spin 14000 rpm 30 seconds.
Discard liquid in collection tube.
Add 200 uL DNA wash buffer
Spin 14000 rpm 1 min.
Discard liquid in collection tube.
Spin 14000 rpm 1 min one more time (dry spin).
Discard collection tube (but not the column).
(Optional: 2nd dry spin into clean collection tube.)
Place column in a clean labeled microtube.
Add 10 uL (min 6 uL for higher DNA concentration) of sterile DDH2O to top of column. Water should be pipetted directly onto center of filter.
Incubate at RT 1 min (or longer).
Spin 1 min at 14000 rpm. Discard the column.
Measure the concentration on the nanodrop. (You may recover the 1uL from the nanodrop if needed.)

Gel Extraction Protocol using QIAquick Gel Extraction Kit:
Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator.
Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV.
Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue.
Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
Add 3 volumes (6 volumes if you are afraid of getting a low yield) of Buffer QG to 1 volume of gel (100mg ~ 100ul)
Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
Add 1 gel volume of isopropanol
Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT)
Run flow-through over column one more time.
After the second time, discard flow-through and place column back in tube.
If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
Add 500uL of Buffer QG to column and centrifuge for 1 min (wash).
Wash: add 0.75ml Buffer PE (make sure that the buffer has ethanol added to it) to column. Let stand for 2-5 minutes and then centrifuge for 1 min
Discard flow-through & centrifuge for 1 min
Place column into clean Eppendorf tube
Add 50ul Buffer EB or water to center of membrane. Make sure to use warm EB (50C). (Use 30uL if worried about low concentration.)
Let stand at RT for 4 min
Centrifuge for 1 min
Measure the concentration using the UV spectrophotometer.
Pro Tips
You don't need 2 lanes if you aren't putting your gel under UV light (the blue light and SYBR safe is fine)
You can up the IPA to 1/4 of the total volume
Warm EB (50 mL conical filled w/ water, plop the tube inside, put it in the heat block)
Don't let it stand at room temperature, you can do it at 5 degrees (heat block)

Gel Extraction Protocol using QIAgen MinElute Kit:
Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator.
Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV.
Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue.
Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 300mg per tube
Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul)
Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
Add 1 gel volume of isopropanol
Add max of 800ul to MinElute column and centrifuge for 1 min (speed >= 10,000 G, RT)
Discard flow-through and place column back in tube.
If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
Add 500 uL of buffer QG and spin column for 1 min and discard flow-through
Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min
Discard flow-through & centrifuge for 1 min
Place column into clean Eppendorf tube
Add 10ul Buffer EB (10 mM TrisCl,pH 8.5) or water to center of membrane
Let stand at RT for 1 min
Centrifuge for 1 min
Measure the concentration using the UV spectrophotometer.

MIT iGEM cookie preparation

Reagents required:
1 1/2 cups (3 sticks) unsalted butter
1 cup sugar
2 large egg yolks
3 3/4 cups sifted all-purpose flour
1/4 teaspoon salt
1 tablespoon pure vanilla extract

Safety note: wear gloves, this procedure involves touching the dough with your hands, a lot. Human tissue is minimum BSL 2 because it can contain contagious pathogens.
Make sure oven is set to 350°F
Melt 3 sticks butter
Add butter and 1C sugar to mixing bowl
mix sugar and butter thoroughly
Add 3.75c all purpose flour
Add .25 tsp salt
Add 1.33Csugar
Add 0.75c cocoa powder
Add 1Tbsp vanilla extract
Add 2 egg yolks
mix until consistent and cohesive, this will likely require kneading.
for all dough
place some amount of dough onto cookie sheet and flatten with palm of hand, should be >3" in diameter and ~.25" thick and flat (no texture/bumps)
press outline cutter into dough and apply pressure around perimeter to ensure a good cut
--The dough should just make it to the angled part of the cookie cutter, if not, adjust height on next cookie
remove cookie cutter
removed excess dough
get more dough and repeat cookie blanking until cookie sheet is full
press stamp into each cookie by pushing on two opposite edges, examine result to see if enough pressure was applied. A cookie can be stamped twice if you're careful.
bake cookies on sheet for 9 minutes
(at this point, running multiple parallel sheets is suggested)
after baking, use a spatula to move cookies onto drying rack
--This is the easiest step to break cookies at, they almost always break teeth off the gears. Make sure that cookies are rotated and aligned such that all gear teeth are supported and they do not fall in between wires on rack.
Wait as long as possible for cookies to cool completely before transferring to a storage container as cookie structural integrity is inversely proportional to temperature. Having a fan blow on cookies to help cooling is suggested.