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.

Samples

  1. 'V' Hair (3 with roots)
  2. 'T' Hair (3 with roots)
  3. 'T' Blood (few μL)

Lane Setup

Lane Setup
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7
Sample λ-HindIII VH VH TH TH TB TB
Primers n/a CSF1PO TPOX CSF1PO TPOX CSF1PO TPOX

Extraction Process

10% Chelex

What I hoped to do
  • 15-30 min room temp
  • 55°C for 30 min
  • Vortex 10s
  • 100°C for 8 min (some debate over 8 v.s 10, went with 8)
  • Spin for a few minutes
What actually happened
  • 15-30 min room temp
  • 55°C for 83 min
  • Vortex 10s
  • 100°C for 8 min
  • Quick spin

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.

PCR notes

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.

  • Primer 1μL each
  • Template 2μL
  • dH2O 10μL
  • Mineral oil 2μL (last)

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)

  • 94°C 2min
  • | 94°C 20s
  • | 59°C 10s
  • | 72°C 30s
  • 72°C 5min

35 cycles "|"

4°C till block reached 4°C (several min), before loading

Total ran about 2 hours I think

Gel notes

2% Agarose TAE (60mL TAE + 1.2g Agarose)

Estimated bp for dyes in 2% gel
XC light blue 750bp
Bromophenol Blue purple 150bp
  • XC contained in PCR mix
  • Bromophenol Blue in λ-HindIII

Let sit in refrigerator till ready (PCR time / few hours)

Electrophoresis

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.

Gel Analysis

2 bands for sample TH did appear (others were empty)! Image to be added after some better processing

Image:07032011-bw.jpg



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