User:Tim Henry/Notebook/Family portrait/2011/07/03
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PCR Take 3
Third attempt at PCR. Previous did not produce any visible results. This one finally worked for one of the three samples.
What I hoped to do
What actually happened
Note: Vortex and Spin were done poor man style here
One protocol mentioned incubating hair for >6 hrs, but blood for 30 mins. So I guess this was a compromise.
V. hair had very little white root visible, whereas T. Hair had a huge white/root section. Did not use Proteinase k for blood, possibly the problem.
Finally remembered to add mineral oil before too late this time. Two caps (equivalent lane 5,7 I think) popped open - closed immediately after, others stayed down. Added some water to wells prior, careful not overflow though. Since tubes don't match thermal cycler I think this is probably necessary.
Next time need to add water first, sit 10min, then pipette to mix bead first. Don't count on boiling to mix it.
Sequences 59, 60, 61 Estimated from cheat sheet and memory (not verified)
35 cycles "|"
4°C till block reached 4°C (several min), before loading
Total ran about 2 hours I think
2% Agarose TAE (60mL TAE + 1.2g Agarose)
Let sit in refrigerator till ready (PCR time / few hours)
50v ~1hr, then 90v (only 80 showing)
Waited until purple reached middle (red band). Didn't want to run too long, since it seems it ran off last time.
1:45 total time
Next time might run 50v for several hours, or 50v till out of wells then 90 immediately after. Usually its really late at this point, hence my impatience.
2 bands for sample TH did appear (others were empty)! Image to be added after some better processing