Daniel:Notebook/HiResChrPaint/2013-7-31
FISH (Started 7/29/2013)[edit]
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]
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
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
So clearly this one didn't work either. I'm going to try another tomorrow, but using the Eppendorf machine.