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PCR
Appending Prefix and SuffixThings to keep in mind!
Annealing temperature of primers (Tm) should be around 60 C
Check the secondary structure of the primers before you order them!
-no individual secondary structures i.e. hairpins
-no heterostructure with the forward and reverse primers together
-free energy of primers should be greater than -4 kCal
-GC content should be around 50% (40-60% is okay)
If using Phusion Master Mix, use this protocol: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531
Dilute your DNA to the following concentrations:
Template:0.1 - 1 ng/ul
Forward Primer:10 uM
Reverse Primer:10 uM
Set up a small box (e.g. empty pipette tip box) with ice and water. Your DNA and polymerase mix will go into this box before going into the the thermocycler in order to limit endonucelase activity.
Add the following DNA to a labeled 0.6ml PCR tube
DNA:Volume
Template:1 uL
Forward Primer:500 nM
Reverse Primer:500 nM
Program the thermocycler as follows
Temperature:Time
98:30s
PAUSE
98:5s
Tm:15
72:(15s)x(#kb)
72:5m
4:forever
Wait for thermocycler to heat up
Add 22.5uL of polymerase mix (Phusion Master Mix) to your DNA. Mix well and spin down. Transfer tubes to ice as soon as possible.
Once the thermocycler has heated up to the right temperature (it should be paused at 98C), add tubes to thermocycler and resume PCR program.
Calculating Reaction Conditions
Use idtdna.com or VectorNTI to calculate melting temperatures of primers
without common overhangs (base pairs 30 to end when read 5' to 3').
PRIMER
Tm
FW
RV
Phusion elongates at a rate of 1kb (1000bp) per 15s. Look up the length of the
gene of interest and calculate time of elongation.
You should get your Tm from NEB.
If you get the melting temperature of your primer from Genious, the annealing
temperature will be that number minus 2.
Assembling Reaction
Get 0.6mL PCR tubes (not the strip tubes).
Get primers for gene of interest. Resuspend if necessary.
Thaw Phusion supermix on ice.
Add the following (in order):
VOLUME:REAGENT
22.5uL (for 35 cycles):Phusion Supermix
2uL:5uM Forward Primer
2uL:5uM Reverse Primer
1uL:Template DNA(~150ng)
Programming The Thermocycler
(In the 3rd floor thermocycler, the PCR program is named "PHUSION")
Initial Denaturation: 98C for 5min
LOOP: 30-35 cycles
CYCLE:
Denaturation: 98C for 10s
Annealing: calculated temperature (typically 55-65C) for 30s
Elongation: 72C for 15s per kb
Final Elongation: 72C for 10min
Store: 4C
MINI PREP
Before you Start:Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000 and mix (If you're the one adding, initial top and check the box on cap)
Add the provided RNase A solution to Buffer P1, mix, and store at 4C. (One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap. Buffer P1 will be in the fridge)
Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume) and then check mark on cap.
Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C Do NOT vortex.
Steps
Pellet 1-5mL of bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature (15-25C; use 2ml microcentrifuge collection tubes). Decant all the liquid and add 1 ml of the culture into the corresponding tube. Make sure not to mix up the tries.
Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to microcentrifuge tube.
Add 250uL Buffer P2 and mix thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture turns blue. Do NOT allow this lysis reaction to proceed for more than 5 min.
Add 350uL of Buffer N3 and mix IMMEDIATELY and thoroughly by inverting tube 4-6 times. Do NOT vortex. Mixture is now colorless.
Centrifuge for 10min at 13,000 rpm in table-top centrifuge.
Apply the supernatant to a QIAprep spin column by decanting or pipetting. Do NOT get any of the sticky precipitate.
Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
Wash the QIAprep column by adding 0.5 mL Buffer PB.
Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
Wash the QIAprep column by adding 0.75 mL Buffer PE.
Centrifuge for 30 - 60s at 13000rpm. Discard flow-through.
Centrifuge for 1 min to remove residual was buffer.
Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50uL Buffer EB to center of each column. Be careful NOT to pierce column.
Let stand for 1 minute.
Centrifuge for 60s at 13000rpm.
Remove column and discard, tube now contains DNA.
Go to NanoDrop and spec DNA.
MIDI PREP
NOTES BEFORE STARTING:Add the provided RNase A solution to Buffer P1, mix, and store at 4C. (One vial of RNase A per bottle of Buffer P1 to give final concentration of 100ug/mL. If you're the one adding, initial top and check box on cap. Buffer P1 will be in the fridge)
Optional: Add LyseBlue reagent to Buffer P2 at a ratio of 1 to 1000 and mix (If you're the one adding, initial top and check the box on cap)
Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume) and then check mark on cap.
Check Buffers P2 and N3 for precipitates, if any redissolve by placing in water bath at 37C. Do NOT vortex.
STEPS:
Harvest bacterial culture by centrifuging at 6000 x g for 15 min at 4C (in 50ml conicals).
Completely resuspend pelleted bacteria in 4ml Buffer P1.
Add 4ml Buffer P2, gently mix by inverting until the lysate appears viscous, and incubate at room temperature (15-25C) for 3 min. If LyseBlue reagent had been added, the cell suspension will turn blue.
Place the QIAfilter Cartridge into a new and suitable tube, allowing space for addition of Buffer BB.
Add 4 ml Buffer S3 to the lysate, and mix by inverting 4-6 times. If LyseBlue reagent has been added, mix the solution until it is completely colorless.
Transfer the lysate to the QIAfilter Cartridge and incubate at room temperature for 10 min. The DNA is now stable; this is an appropriate place in the protocol to pause (if necessary).
During incubation, place QIAGEN plasmid Plus spin columns into the QIAvac 24 Plus. Insert Tube Extenders into each column.
Gently insert the plunger into the QIAfilter Cartridge and filter the cell lysate into the tube.
Add 2 ml Buffer BB to the cleared lysate, and mix by inverting 4-6 times.
Transfer lysate to a QIAGEN Plasmid Plus spin column on the QIAvac 24 plus.
Apply approximately -300 mbar vacuum until the liquid has been drawn through all columns.
To wash DNA, add 0.7 ml Buffer ETR and apply vacuum until the liquid has been drawn through all columns.
To further wash the DNA, add 0.7 ml Buffer PE and apply vacuum until the liquid has been drawn through all columns.
To completely remove the residual wash buffer, centrifuge the column at 10,000 x g (9,700 rpm) for 1 min in a tabletop microcentrifuge.
Place the QIAGEN Plasmid Plus spin column into a clean 1.5 ml tube. To elute the DNA, add 200 ul Buffer EB or water to the center of the QIAGEN Plasmid Plus spin column, let it stand for > 1 min, and centrifuge for 1 min.
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 TipsRun 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 joinedUse 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)