Byte Amplification and End Preparation

This procedure allows for the amplification of any gene or part in the pAB and pBA plasmids. The procedure is the same for both pAB and pBA Byte Amplifcation with the only difference being the universal primers (for pAB Bytes use pAB+ and pAB-, for pBA Bytes use pBA+ and pBA-).

What you will need:

• Overnight with bacterial growth
• Screw cap tubes
• Glycerol

Byte PCR and Digestion:

1. In an PCR Tube mix the following (Assuming a 100 μL Reaction Volume):

• 4 μL Template (pAB or pBA – 1.5 ng/μL)
• 4 μL Forward Universal Primer (10 μM)
• 4 μL Reverse Universal Primer (10 μM)
• 2 μL PFU Cx (2.5 U/μL)
• 10 μL PFU Cx Buffer (10X)
• 10 μL dNTPs (2mM)
• 66 μL ddH2O

2. Run the PCR reaction under the following conditions:

• 95˚C (2 Minutes)
• 30 Cycles of:
  1. 95˚C (30 Seconds)
  2. 56˚C (1 Minute)
  3. 72˚C (2 Minutes)
• 72˚C (5 Minutes)
3. Add 1 μL USER™ (New England Biolabs) to each PCR tube and incubate at 37˚C for 1 hour. 4. Run the PCR product on an agarose gel. 5. Gel column purify to isolate PCR product (add the 10 uL 3M sodium acetate prior to loading onto column) 6. (ethanol precipitation may be required).

Ethanol Precipitation

• If we make extra we have them if we need to send them out or want to store in several locations.