Julius B. Lucks/Protocols

LB/LB Agar

• 5.0 g Bacto Tryptone (NOT peptone)
• 2.5 g Yeast Extract LD
• 5.0 g NaCl

1. Add ingredients to 500 ml H20.
2. For LB Agar, add 7.5 g Bacto Agar after ingredients dissolve.
3. Autoclave

Note: Difco brand preferred

BNMS

H20:BN:BSA::5:2:2

Glycerol Stocks

• 1/2 ml o/n culture
• 1/2 ml 50% sterile glycerol in H20

1. Place in cryovial and store in -80 freezer

Agarose Gels

2% Stock

• 4g Agarose
• 196 mL TAE

1. Place in bottle and heat gently until dissolves

1% Gels

• 25mL TAE
• EtBr
• 25mL 2% Agarose/TAE

1. Combine, mix gently, pours 2 small gels

Transformations

• Each aliquot (200 uL) of competent cells can transform 4 samples.

1. Get appropriate aliquots of competent cells from -80 and thaw on ice (about 15 minutes).
   • Also get KCM from fridge and put on ice.
2. Take an aliquot and add 30 uL KCM - mix gently with pipette.
3. Add 50 uL of the Cells+KCM mix to the appropriate tube, close and put on ice.
4. Once all tubes have been given cells, incubate on ice for 20 minutes.
5. Heat shock tubes by placing in 42 C plate for 1.5 minutes.
6. Place tubes back on ice.
7. If using Amp, plate.
8. If using other antibiotics, add 50 uL 2 YT rich media and place tubes at 37 C, 200 RPM for 1 hour.
9. Plate.

Large Experiment Transformations

1. Aliquot plasmids into 96-well pcr plates
2. Add 30 uL cells w/o KCM
3. Incubate on ice for 20 min
4. Heat shock in water bath at 42C for 1.5 mins
5. Transfer cells to 96-well block (1ml) with 50uL 2YT media in appropriate wells
6. Recover in Vortemp at 37C, 70 RPM for 1 hr
7. Plate using glass beads

Phusion PCR

λ         Ingredients
----------------------------
37               H2O
10               Phu Buff 5X
1                 dNTPs 
0.5             Forward primer
0.5             Reverse primer
0.5             template DNA            
0.5             Phusion
----
50

1. Combine ingredients in PCR tube in that order, perform following PCR program
   • 1 = 98 C for 0:30
   • 2 = 98 C for 0:15
   • 3 = T C for 0:30 (Where T = 3C above the lowest Tm primer)
   • 4 = 72 C for target_length_kB*15s (15s/kB)
   • 5 = GOTO 2 24 TIMES
   • 6 = 72 C for 5:00 (or larger if step 4 > 5:00)
   • 7 = 4 C for ever
   • 8 = END

Note: I have found through optimization tests and looking at the NEB troubleshooting guide that 15s/kB extension and running for 25 cycles can reduce side reactions. To further reduce side reactions, Phusion Hot Start can be used instead of Phusion with the same protocols. Phusion Hot Start has an antibody bound to it that prevents it from working at room temperature. The antibody unbinds during the first 98C step. This technique is especially useful for eliminating side reactions due to primer dimers.

Phusion PCR with Phosphorylated Oligos

λ         Ingredients
----------------------------
28               H2O
10               Phu Buff 5X
1                 dNTPs 
5               Forward primer
5               Reverse primer
0.5             template DNA            
0.5             Phusion
----
50

1. Follow same PCR program as Phusion PCR

DpnI Digest

(Note this probably uses too much enzyme. Need to test with lower enzyme levels.)

λ         Ingredients
----------------------------
14.25         H2O
2.5            10x Buffer 4
7.5           PCR solution 
0.75          DpnI
-----
25

1. Combine ingredients in this order 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.

Oligo Phosphorylation

Ingredients:

• 1 uL oligo (100 uM stock)
• 1 uL 10x T4 ligase buffer (black striped aliquat in freezer, contains ATP)
• 7.5 uL water
• 0.5 uL PNK (enzyme freezer - modifying enzyme T4 PNK) (NEB M0201L)

1. Combine ingredients (one tube for each primer) in a tube and incubate at 37 C for 1 hr.

Quick Ligation

See NEB Technical Bulletin 2000 for more information.

Ingredients:

• DNA
• H20
• 10 uL 2x Quick Ligation Buffer
• 1 uL Quick Ligase

1. Added 50-150ng DNA to a tube (I usually use 3 uL of a gel purified PCR product. Note: It is much preferred to PCR purify to allow nanodrop quantification of the product so that the precise amount of solution to give 150ng of DNA for the ligation can be used. Due to a compound in Buffer QG interfering with the spectra taken by the nanodrop, this normally cannot be done when the product is run on a gel and then gel purified with a Qiagen Kit. The PCR purification kit does not interfere with nanodrop quantification and is hence preferred. Anecdotally, a Gel purification followed by a PCR purification will remove the compound in Buffer QG and allow nanodrop quantification.)
2. Add enough H20 for a final volume of 10 uL
3. Add ligation buffer
4. Add Quick Ligase
5. Incubate at RT for 5 min

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

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 recording 260/280, 260/230 and ng/uL.

1. • make sure 260/280 and 260/230 are > than 1.8, and ng/ul higher than 30

1. 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

1. 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).
2. 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.
3. Incubate 1.5 minutes per basepair of overlap at room temp (If 10bp homology, 15 minutes incubation).
4. Add 1/10 volume of 10 mM dCTP to each tube.
5. Mix the content of the tubes at the correct ratio (use second table below).
6. Add 1/10 of the volume of 10X T4 DNA ligase buffer.
7. Incubate 30 minutes at 37C.
8. 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.

iPCR

(This is basically the same procedure as NEBs Site-Directed Mutagenesis, or 'Round-the-horn_site-directed_mutagenesis).

Once primers are designed for addition, deletion, point mutations, etc. Perform the following:

1. Oligo Phosphorylation
2. Phusion PCR with Phosphorylated Oligos
3. Gel Purification
4. DpnI Digest
5. Quick Ligation
6. Transformation

The whole process takes about a day.

96 Well Format Experiment

• Use 2mL 96 well blocks
• Use Vortemp shakers

1. 400 uL LB + antibiotics in wells
2. Pick colonies with 200 uL pipette tips and place in wells
3. gently shake block by hand
4. use multi channel pipette to pipette tips up and down 5 times
5. remove tips
6. cover block (can let sit at RT on bench all day)
7. grow at 37 C, 70 RPM o/n
8. add 237 uL MM + antibiotics + inducers to fresh block
9. innoculate new block with 13 uL of o/n cultures
10. grow at 37 C, 70 RPM (usually 4 hrs)
11. take measurement with 100 uL MM + 100 uL 2 mg/mL Kan in PBS

Mixed Oligo Codes

• R - A,G
• Y - C,T
• M - A,C
• K - G,T
• S - C,G
• W - A,T
• H - A,C,T
• B - C,G,T
• V - A,C,G
• D - A,G,T
• N - A,C,G,T