Daniel:Notebook/HiResChrPaint/2013-7-31

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FISH (Started 7/29/2013)[edit]

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FISH Slide Preparation[edit]

Following original FISH protocol

Day 2

10. The next day, remove coverslip carefully and wash slides in pre-warmed jar containing 2X SSCT at 60C for 15 minutes
11. Incubate slides in 2X SSCT at room temperature for 10 minutes
12. Incubate slides in 0.2X SSC at room temperature for 10 minutes
13. Tap slides dry on papertowels; do not let the surface containing the cells touch the paper towels
14. Add 15 uL mounting media such as SlowFadeGold with DAPI to a 22x30 #1.5 coverslip
15. Invert slides onto coverslips with mounting media
16. Seal each slide using nail polish
17. Allow at least 30 minutes for nail polish to dry
18. Slides are quite stable for several months at 4C

S1 Probe Preparation[edit]

Making a new batch of S1 probe, since I have the other three. Yesterday's didn't work, but considering the results, it's most likely that I didn't add a reagent. So I guess I need to be more careful this time.

PCR Amplification[edit]

1. Reaction Table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.2 50 0.4 0.4 0.4 0.4 2.5 45.7 100
V4S1 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S1 (12.5x) 2.5 625 0 0 5 5 31 581.5 1250
2. Aliquot 100 uL into each of 12 qPCR tubes 
3. PCR protocol:
    i. PCR 3 min 95C
    ii. 95C 3 sec
    iii. 55C 30 sec 
    iv. 72C 20 sec
    v. plate read
    vi. goto ii x17
    vii. 72C 2 min
    viii. 16C hold

qPCR Results[edit]

File:2013-7-31-S1.png

So this didn't work again. Time for troubleshooting.

Troubleshooting qPCR[edit]

TBE Gel[edit]

Checking the template DNA using a TBE gel. I'm running 1,2, and 3 for a check.


Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 25bp Ladder Probe Set 1 Probe Set 2 Probe Set 3

Gel Image

File:ProbeTest 073113.jpg

PCR (Noi)[edit]

Noi ran PCR on S1 and S2. She found they amplified as usual. Two things were different in Noi's run: (1) she used the other thermocycler (I always use Bio-Rad) and (2) she used different KAPA mix.

Column Purification[edit]

I went ahead and column purified 3 of the PCR reactions for V4 and V6 (condensed to 1 column each).

Results:

  ng/uL ug in 40 uL
V4S1 5.5 0.2
V6S1 5.2 0.2

So, summing up the results: (1) the template DNA is at least there. (2) PCR using a different thermocycler and a different SYBR fast master mix works just fine. (3) My PCR did in fact fail (3 confirms that the Bio Rad plate reader is still working).

So my guess is that the master mix is not working correctly.

PCR (Me)[edit]

Since I'm pretty sure that the PCR is working fine, except that the master mix I used wasn't working, I'm going to try again (2 reactions only!) and see if I can't PCR something, one using my MM and one using Noi's.

  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
V4S1D 0.5 50 0.5 0.5 0.5 0.5 0 47.5 100
V4S1N 0.5 50 0.5 0.5 0.5 0.5 0 47.5 100

qPCR Results

File:2013-7-31-S1Test.png

So clearly this one didn't work either. I'm going to try another tomorrow, but using the Eppendorf machine.