Daniel:Notebook/HiResChrPaint/2013-8-1: Difference between revisions

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=FISH=
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
Hosuk and I went to the microscope room today.  We got a few images that might be worthwhile.  We also looked closer at the microscopes used by Beliveau et al, and we realized that two of the microscopes they mention are almost exactly the same models we use.  One matches the confocal in the basement, the other matches the Olympus in 406.  So I can and should use the Olympus from now on to screen for good results.
==Troubleshooting qPCR ([[Daniel:Notebook/HiResChrPaint/2013-7-31#Troubleshooting qPCR|Started 7/31/2013]])==
==Troubleshooting qPCR ([[Daniel:Notebook/HiResChrPaint/2013-7-31#Troubleshooting qPCR|Started 7/31/2013]])==
[[Danie:Notebook/HiResChrPaint|Back to Calendar]]


===PCR 2 (Me)===
===PCR 2 (Me)===


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.
As per yesterday's finale, I'm trying single reaction PCR again, comparing Noi and my Master Mixes, and using the Eppendorf thermocycler instead of the BioRad one.


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
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|}
|}
====qPCR Results====
[[Image:2013-8-1-ProbeTest_S1.png|600px]]
===PCR 3 (Water Test?)===
So now it would seem the only avenue left to check is the purity of the water I've been using.  I don't know why this would be the problem but there it is.  Here's the setup:
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center" valign="bottom"
| align="center" width="65" height="45" | &nbsp;
|style="font-weight:bold" width="65" | 20 nM OligoPool
|style="font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM
|style="font-weight:bold" width="65" | 100 uM AP1V41U
|style="font-weight:bold" width="65" | 100 uM AP2V4
|style="font-weight:bold" width="65" | 100 uM AP1V61U
|style="font-weight:bold" width="65" | 100 uM AP2V6
|style="font-weight:bold" width="65" | 2 mM aa-dUTP
|style="font-weight:bold" width="65" | H20
|style="font-weight:bold" width="65" | Total Volume
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | V4S1D
| align="center" | 0.5
| align="center" | 50
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0
| align="center" | 47.5
| align="center" | 100
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | V4S1D*
| align="center" | 0.5
| align="center" | 50
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0
| align="center" | 47.5
| align="center" | 100
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | V4S1N*
| align="center" | 0.5
| align="center" | 50
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 0
| align="center" | 47.5
| align="center" | 100
|}
====qPCR Results====
[[Image:2013-8-1-S1-WaterTest.png|600px]]
So yeah, looks like that was the problem.  The green line is the sample that used the old water sample.  Guess I'm throwing away that bottle of water, and I'm going to use aliquots in the future to help reduce contamination.

Latest revision as of 19:17, 5 August 2013

FISH[edit]

Back to Calendar

Hosuk and I went to the microscope room today. We got a few images that might be worthwhile. We also looked closer at the microscopes used by Beliveau et al, and we realized that two of the microscopes they mention are almost exactly the same models we use. One matches the confocal in the basement, the other matches the Olympus in 406. So I can and should use the Olympus from now on to screen for good results.


Troubleshooting qPCR (Started 7/31/2013)[edit]

PCR 2 (Me)[edit]

As per yesterday's finale, I'm trying single reaction PCR again, comparing Noi and my Master Mixes, and using the Eppendorf thermocycler instead of the BioRad one.

  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[edit]

File:2013-8-1-ProbeTest S1.png

PCR 3 (Water Test?)[edit]

So now it would seem the only avenue left to check is the purity of the water I've been using. I don't know why this would be the problem but there it is. Here's the setup:

  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
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[edit]

File:2013-8-1-S1-WaterTest.png

So yeah, looks like that was the problem. The green line is the sample that used the old water sample. Guess I'm throwing away that bottle of water, and I'm going to use aliquots in the future to help reduce contamination.