Julius B. Lucks/Protocols

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

1. 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
----------------------------
5            10x Buffer 4
1.5          DpnI
28.5         H2O
15           PCR solution 
-----
50

1. 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)

1. Combine ingredients (one tube for each primer) in a tube and incubate at 37 C for 1 hr.
2. Combine the 2 tubes, and add 180 uL water
3. 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)
4. 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)

1. Design primers to amplify pieces of interest so that the extremities contain 10-40 bp of homology with the target region
2. 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.
3. Prepare 10mM dCTP (NEB dCTP: 100mM dilute by 10X with H2O)
4. Prepare 0.5U/ul T4 DNA polymerase (NEB T4 DNA polymerase comes 3U/ul, so dilute by 6X with H2O)
5. Quantify your products using a nanodrop.
   • make sure 260/280 and 260/2 higher 30 are both than 1.8
6. 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.
7. Aliquot 50-100ng 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 2:1 molecular ratio relative to the backbone (volume doesn't matter). We have done 5:1 with a modicum of success.
8. 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.
9. Incubate 1.5 minutes per basepair of overlap at room temp (If 10bp homology, 15 minutes incubation).
10. Add 1/10 volume of 10 mM dCTP to each tube.
11. Mix the content of the tubes at the correct ratio.
12. Add 1/10 of the volume of 10X T4 DNA ligase buffer.
13. Incubate 30 minutes at 37C.
14. 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
----    --------    ------    ------    ---------------------    --------    ------
___
___
___

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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.