Griffitts:Overlap-extension PCR

Overlap Extension PCR

Introduction

This procedure is used to generate a construct for making an in-frame deletion of a gene. It involves first amplifying DNA at either end of the target gene. The inside primers are designed to overlap each other so that in the second round of PCR the two can be zipped together. The resulting clone will then be ligated into pJQ200SK for deletion of the target gene.

Round One

• Prepare your template sample
  • For amplifying Sinorhizobium sequences, boil a dense suspension of cells in 35 μL PCR lysis buffer for ~2 min and vortex
  • For amplifying E. coli, adding cells directly to the reaction is sufficient
• Thaw the Pfx50 buffer, dNTPs, and primers
  • Keep Pfx50 polymerase on ice throughout the procedure
• Combine components according to the First Round recipe below
  • Set the reaction up on ice
  • You will need to set up separate reactions for amplifying the upstream fragment and the downstream fragment
• Select the appropriate cycling program and verify that all of the parameters are correct
  • Allow 1 minute of extension time per 1 KB DNA being amplified
• Select "Run program" and select "YES" when asked about heated lid
• Wait until block has reached the beginning temperature then place PCR tubes into the machine
• Lower the lid until it latches and slightly tighten the heated lid onto the tubes

NOTE: When the PCR machine has finished cycling, your samples may be stored at –20°C. You may now proceed to gel electrophoresis and/or PCR clean-up.

Round One recipe

Per reaction	Ingredient
27.4 μL	dH2O
4.0 μL	10X Pfx buffer
1.2 μL	dNTPs
1.0 μL	Pfx polymerase
1.6 μL	DNA template
2.4 μL diluted 1:10	forward primer
2.4 μL diluted 1:10	reverse primer

Round One cycling

Step	Temp	Duration
1	94.0°C	2:00
2	94.0°C	0:20
3	55.0°C	1:00
4	70.0°C	1:00
5	GOTO 2	35 times
6	70.0°C	3:00
7	4.0°C	forever
8	END

Round Two

• Thaw the Pfx50 buffer, dNTPs, and primers
  • Keep Pfx50 polymerase on ice throughout the procedure
• Combine components according to the Second Round recipe below
  • Set the reaction up on ice
  • Be sure to use cleaned-up DNA from the first round reactions
• Select the appropriate cycling program and verify that all of the parameters are correct
  • Allow 1 minute of extension time per 1 KB DNA being amplified
• Select "Run program" and select "YES" when asked about heated lid
• Wait until block has reached the beginning temperature then place PCR tubes into the machine
• Lower the lid until it latches and slightly tighten the heated lid onto the tubes

NOTE: When the PCR machine has finished cycling, your samples may be stored at –20°C. You may now proceed to gel electrophoresis and/or PCR clean-up.

Round Two recipe

Per reaction	Ingredient
27 μL	dH2O
4.0 μL	10X Pfx buffer
1.2 μL	dNTPs
1.0 μL	Pfx polymerase
1.0 μL	Upstream DNA fragment clone
1.0 μL	Downstream DNA fragment clone
2.4 μL diluted 1:10	forward primer
2.4 μL diluted 1:10	reverse primer

Round Two cycling

Step	Temp	Duration
1	94.0°C	1:00
2	94.0°C	0:25
3	40.0°C	0:25
4	70.0°C	1:20
5	GOTO 2	4 times
6	94.0°C	0:25
7	55.0°C	0:25
8	70.0°C	1:20
9	GOTO 6	26 times
10	70.0°C	1:00
11	4.0°C	forever
12	END

Primer Dilution

• 27 μL dH₂O
• 3 μL primer

Repeat for each primer