Julius B. Lucks/Protocols
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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
λ Ingredients ---------------------------- 20 B2MS 2.5 DpnI 2.5 H2O 25 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)
- Design primers to amplify pieces of interest so that the extremities contain 20-40 bp of holomoly 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.
- make sure 260/280 and 260/230 are both higher than 1.8
- Aliquot 50-100ng of your backbone in a new tube. Put the other pieces in separate tubes, equivalent to a 2:1 molecular ratio relative to the backbone (volume dosen't matter).
- Add 1/10 of the volume of 10X T4 DNA ligase buffer in each tube
- Add 1 ul 0.5u/ul T4 DNA polymerase to each tube.
- Incubate 30 minutes at room temp.
- Add 1/10 volume of 10 mM dCTP to each tube.
- Mix the content of the tubes. Incubate 30 minutes at 37C.
- Transform E. coli competent cells.
Tube Concentration (ng/ul) Aliquot (ul) Vol 10x ligase buffer Vol T4 Pol Total Vol Vol dCTP
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