PCR with ROCHE Expand™ High Fidelity PCR System

Principle and Notes:

• Expand High Fidelity PCR System is a mixture of Taq DNA polymerase and a DNA polymerase with proofreading activity for high yield and fidelity. It is optimized for PCR of DNA fragments up to 5 kb. Great for amplifying HIV Env

• Use 2.6 U for a standard 50 μl PCR.

• Principle: We are making 2 mixes

• Mix#1: contains dNTP primers and template (are specific of what need to be amplified)

• Mix#2 contains the buffer and polymerase

Materials and Reagents

• EHIFI-RO ROCHE Expand™ High Fidelity PCR System

• dNTPs

Equipment

• PCR machine

Procedure

1. Dilute DNA template (DNA template ~0.1-250 ng/ul

2. Dilute primers at 20 pmol/µl=20μM

3. Prepare mix#1 directly in the PCR tube. In each tube, add:

        H20_________________________ 21 µl

        dNTP (10mM)__________________ 1 μl

        Primer antisense (20 µM)_________ 1 μl

        Primer sense (20 µM)____________ 1 μl

        Template (0.1 to 250ng)___________1μl

4. Prepare Master mix #2 (25ul per sample, can be prepared as master mix)

        H20________________________. 19.5 µl

        Buffer 2 (10X)__________________ 5 μl

        Taq__________________________ 0.5 μl

5. Vortex briefly to mix the Mix#2

6. Add 25ul of Mix#2 to the PCR tubes containing already Mix#1

7. PCR cycle

            94˚C 2min

            (94˚C 30s, 58˚C 30s, 72˚C 3 min) *30

            72˚C 30 min (if subcloning into TOPO vectors)

Used in:

Enhanced fusion and virion incorporation for HIV-1 subtype C envelope glycoproteins with compact V1/V2 domains. Cavrois M, Neidleman J, Santiago ML, Derdeyn CA, Hunter E, Greene WC. J Virol. 2014 Feb;88(4):2083-94. doi: 10.1128/JVI.02308-13. Epub 2013 Dec 11.

Measuring HIV fusion mediated by envelopes from primary viral isolates. Cavrois M, Neidleman J, Galloway N, Derdeyn CA, Hunter E, Greene WC. Methods. 2011 Jan;53(1):34-8. doi: 10.1016/j.ymeth.2010.05.010. Epub 2010 Jun 8