General PCR Protocol

See also: <a href="https://docs.google.com/document/d/16Piw2IQOHUwHPN0zY0PJp5cm4c9gFu6VLiOj-1kspP8/edit?usp=drive_web">PCR_GoTaq</a> and <a href="https://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq(R) DNA Polymerase Product Information Sheet ... - Promega</a>

Resuspending primers:

<a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">protocol: resuspending PCR primers</a>

Working Stock:

(serves as a backup)

Dilute to 10 times the number on primer vial

10ul fwd primer 90ul DI water

10ul rev primer 90ul DI water

Notes for iGEM 2013 HSB lab:

We are using the GreenTaq polymerase 2X pre-mixed (<a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>) for PCR.

Use the protocols below for Green Taq polymerase.

Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).

If PCRing insert use 1ng/ul DNA concentration.

Amplification PCR with Green Taq polymerase:

1. Setup Amplification PCR Reaction

1. 1uL Template DNA

2. 22ul deionized (dI) H2O

3. 1ul 10uM forward primer (Tm XX C)*

4. 1ul 10uM reverse Primer (Tm YY C)*

5. 25ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)

6. Total 50ul

2. Amplification PCR Reaction Program

1. 95 C for 30s

2. 95 C for 30s

3. ZZ* C for 30s (important step)

4. 72 C for 1min per kb target gene plus 10-15% (important step)

5. Repeat 2-4 34x

6. 72 C for 5 to 10min

7. 4 C for forever (don’t forget to turn the machine off the next day)

• Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).

Analytical PCR with Green Taq polymerase:

3. Setup PCR Reaction

1. 1uL Template

2. 2ul dI H2O

3. 1ul 10uM forward primer (Tm XX C)*

4. 1ul 10uM reverse Primer (Tm YY C)*

5. 5ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)

6. Total 10uls

4. PCR Reaction Program

1. 95 C - 30s

2. 95 C - 10s

3. ZZ* C - 10s (important step)

4. 72 C - 1min per kb target gene plus 10-15% (important step)

5. Repeat b-d 25x

6. 72 C - 5 to 10min

7. 10 or 4 C - forever

Phusion polymerase:

5. Setup Amplification PCR Reaction

1. 1uL Template

2. 1uL 25mM dNTP's

3. 10uL Phusion HF Buffer

4. 1.0ul Forward Primer (Tm XX oC)*

5. 1.0ul Reverse Primer (Tm YY oC)*

6. 0.5uL Phusion polymerase

7. 36.5uL diH2O

8. Total 50uls

6. Amplification PCR Reaction Program

1. 98 C - 30s

2. 98 C - 10s

3. ZZ* C - 10s (important step)

4. 72 C - 30s per kb target gene plus 10-15% (important step)

5. Repeat b-d 29-34x

6. 72 C - 5 to 10min

7. 10 or 4 C - forever

• Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).

Refer for further details to NEB's online protocol for Phusion:

<a href="http://www.neb.com/nebecomm/products/protocol87.asp">http://www.neb.com/nebecomm/products/protocol87.asp</a>