Julius B. Lucks/Protocols
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Glycerol Stocks
- 1/2 ml o/n culture
- 1/2 ml 50% sterile glycerol in H20
- Place in cryovial and store in -80 freezer
Agarose Gels
2% Stock
- 4g Agarose
- 196 mL TAE
- Place in bottle and heat gently until dissolves
1% Gels
- 25mL TAE
- EtBr
- 25mL 2% Agarose/TAE
- Combine, mix gently, pours 2 small gels
Transformations
- Each aliquot (200 uL) of competent cells can transform 4 samples.
- Get appropriate aliquots of competent cells from -80 and thaw on ice (about 15 minutes).
- Also get KCM from fridge and put on ice.
- Take an aliquot and add 30 uL KCM - mix gently with pipette.
- Add 50 uL of the Cells+KCM mix to the appropriate tube, close and put on ice.
- Once all tubes have been given cells, incubate on ice for 20 minutes.
- Heat shock tubes by placing in 42 C plate for 1.5 minutes.
- Place tubes back on ice.
- If using Amp, plate.
- If using other antibiotics, add 50 uL 2 YT rich media and place tubes at 37 C, 200 RPM for 1 hour.
- Plate.
Phusion PCR
λ Ingredients ---------------------------- 37 H2O 10 Phu Buff 5X 1 dNTPs 0.5 template DNA 0.5 Phusion 0.5 Forward primer 0.5 Reverse primer ---- 50
- Combine ingredients in PCR tube, perform following PCR program
- 1 = 98 C for 0:30
- 2 = 98 C for 0:15
- 3 = 55 C for 0:30
- 4 = 72 C for target_length_kB*20s (20s/kB)
- 5 = GOTO 2 30 TIMES
- 6 = 72 C for 2:00 (or larger if step 4 > 2:00)
- 7 = 4 C for ever
- 8 = END
DpnI Digest
(Note this probably uses too much enzyme. Need to test with lower enzyme levels.)
λ Ingredients ---------------------------- 5 10x Buffer 4 1.5 DpnI 28.5 H2O 15 PCR solution ----- 50
- Combine ingredients and incubate at 37 C for 1 hr.
Pre-Cut Oligo Phosphorylation
Ingredients:
- 1 uL primer
- 1 uL 10x ligase buffer (black striped aliquat in freezer)
- 7.5 uL water
- 0.5 uL PNK (enzyme freezer - modifying enzyme T4 PNK)
- Combine ingredients (one tube for each primer) in a tube and incubate at 37 C for 1 hr.
- Combine the 2 tubes, and add 180 uL water
- Take this tube and boil for 5 min on a hot plate in a beaker of water (boil water in a beaker and put the tube in the boiling water)
- Take beaker off hot plate and put on bench to ramp down to room temperature
Use this solution as you would for an insert in a ligation.
Glucose Minimal Media
- Ammonium Sulfate (NH4)2SO4 - 0.502g (0.0038 mol)
- MW 132.14 g/mol
- CAS 7783-20-2
- Dibasic Potassium Phosphate KH2PO4*3H2O - 2.244g (0.01 mol)
- MW 228.23
- CAS
- Monobasic Potassium Phosphate K2HPO4 - 5.226g (0.038 mol)
- MW 136.09
- CAS 7778-77-0
- Sodium Citrate - Na3C6H5O7 - 0.25g (0.00085 mol)
- HOC(COONa)(CH2COONa)2*2H2O
- MW 294.10
- CAS
- Dextrose Anhydrous (glucose) - 1.0g (0.0056 mol)
- CH2OH(CHOH)4CHO
- MW 180.16
- CAS 50-99-7
- Thiamine (0.1% stock) - 0.25ml (xxx mol)
- MW
- CAS
- Magnesium Sulfate MgSO4*7H2O (1M stock) - 0.5ml (0.0005 mol)
- MW 246.48
- CAS 10034-99-8
- H2O - up to 0.5L
Glycerol Minimal Media
Same as Glucose Minimal Media except substitute for Dextrose Anhydrous
- Glycerol C3H8O3 (50% stock) - 1.04 ml (0.52 g = 0.0056 mol)
- MW 92.09
- CAS 56-81-5
SLIC
Reference : Li and Elledge, Nature Methods (2007)
Pre-SLIC
- Design primers to amplify pieces of interest so that the extremities contain 10-40 bp of homology with the target region
- Amplify pieces with Phusion PCR. Note: gel purify or DpnI digest your products if you used a template with the same antibiotic resistance as your target molecule; otherwise, PCR purification columns are fine. Pieces (e.g.vector backbone) can also be prepared by digestion.
- If the PCR piece is around the same size as the full plasmid, DpnI digest is recommended.
- Prepare 10mM dCTP (NEB dCTP: 100mM dilute by 10X with H2O)
- Prepare 0.5U/ul T4 DNA polymerase (NEB T4 DNA polymerase comes 3U/ul, so dilute by 6X with H2O)
- Quantify your products using a nanodrop recording 280/260, 280/230 and ng/uL.
- make sure 260/280 and 260/2 higher 30 are both than 1.8
- If you are planning a DpnI digest, make dilutions of the backbone. We have found that digests of PCR purification elutions do not digest completely. 1:20 dilutions appear to be better. That's 1.5ul purification in 28.5ul water, and then 15ul of that into a DpnI digest.
SLIC Reactions
- Using first table below, aliquot 100ng of your backbone in a new tube (For dilutions above 1:20, where the nanodrop fails to quantify backbone, I do half the DpnI digestion). Put the other pieces in separate tubes, equivalent to a 5:1 molecular ratio relative to the backbone (volume doesn't matter).
- Add 1 ul 0.5u/ul T4 DNA polymerase to each tube. This includes the insert if it is a PCR result, but not if it is two oligos annealed.
- Incubate 1.5 minutes per basepair of overlap at room temp (If 10bp homology, 15 minutes incubation).
- Add 1/10 volume of 10 mM dCTP to each tube.
- Mix the content of the tubes at the correct ratio (use second table below).
- Add 1/10 of the volume of 10X T4 DNA ligase buffer.
- Incubate 30 minutes at 37C.
- Transform E. coli competent cells.
Tube Conc (ng/ul) Aliquot (ul) Vol T4 Pol Total Vol Vol dCTP Total Vol ------- ------------ ------------ ---------- --------- -------- --------- ___ ___ ___ Tube Backbone Insert Volume Vol 10x ligase buffer Colonies Picked ---- -------- ------ ------ --------------------- -------- ------ ___ ___ ___
Julius B. Lucks 19:34, 4 August 2008 (UTC): General note - we have been using PCR -> PCR purification (nanodrop and run a small amount on a gel at this point) -> dilute 20x with H2O -> use this dilution to purform DpnI digests -> use these digests directly in the SLIC reaction as described above.
- Note that if the PCR is not 'pure' (large primer bands or alternative products) then the nanodrop quantification will be off.