CHP
The DNA is put in as a phage from which (if I can) I filter out the bacteria.
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1 ul |
1:5 dilution of a 1x10E11 phage stock |
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1 ul |
polymerase enzyme (Boehringer Mannhein Expand High Fidelity, which has as high fidelity as PFU, but we found we got a higher yield. |
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10 ul |
PCR buffer (we use the higher [Mg]: 15mM) |
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10 ul |
F primer (10uM) to give 1uM, which is a 1/100 dilution of the master primer mix |
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10 ul |
rev. primer; same concentration as F primer |
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0.8 ul |
100mM dNTP mix (25mM of each) Stratagene cat.# 200415-52 |
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67.2 ul |
H2O (to 100 ul) |
Mix the buffer, primers, dNTP and water, then add enzyme. Mix gently and distribute to 0.5 ml tubes (not necessarily PCR tubes). Then add the source of the DNA (phage). Spin briefly to bring liquid to bottom of tube, then I add a wax bead on top (I do not use mineral oil).
Ensure you have a control reaction: a PCR mix without the DNA source.
I put this in the thermocycler with a cooling step at the end:
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I run out 5 ul of sample with 1ul of sample buffer on a 1% agarose gel to check if the amplification worked and if the control is clean.
Clean up using the Boehringer Mannheim's "High Pure PCR Product Purification Kit" (you may lose some DNA but the final purity is high).
Examine the resulting cleaned up DNA by agarose gel to determine the amount of DNA. There may be DNA in background; however, this does not affect manual sequencing.