Daniel:Notebook/HiResChrPaint/2013-10-3: Difference between revisions
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The control sample worked well enough, it seems like there is plenty of signal but it isn't in any particular place. The experiment group, however, looked terrible, as it looked like there were no cells anywhere on the dish. Further, there were scratches everywhere, distorting the view. I'll have to make sure on future samples that the cells, after fixation, look healthy and plentiful. | The control sample worked well enough, it seems like there is plenty of signal but it isn't in any particular place. The experiment group, however, looked terrible, as it looked like there were no cells anywhere on the dish. Further, there were scratches everywhere, distorting the view. I'll have to make sure on future samples that the cells, after fixation, look healthy and plentiful. | ||
=V6S3/V4S1 Probe Production (Amino primers)= | |||
Since I'm out of V6S3 and V4S1 I'm going to make some more. | |||
==qPCR Amplification== | |||
1. Reaction Table | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#FFFF00" align="center" width="65" height="45" | | |||
| width="65" | 20 nM OligoPool | |||
| width="65" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM AP1V41U-Amino | |||
| width="65" | 100 uM AP2V4 | |||
| width="65" | 100 uM AP1V61U-Amino | |||
| width="65" | 100 uM AP2V6 | |||
| width="65" | H20 | |||
| width="65" | Total Volume | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
| height="30" | std 100 uL rxn | |||
| align="center" | 0.2 | |||
| align="center" | 50 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 0.4 | |||
| align="center" | 46.5 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" | |||
| height="30" | V4S1 (12.5x) | |||
| align="center" | 2.5 | |||
| align="center" | 125 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 581.25 | |||
| align="center" | 1250 | |||
|- style="font-size:12pt" align="center" | |||
| height="30" | V6S3 (12.5x) | |||
| align="center" | 2.5 | |||
| align="center" | 125 | |||
| align="center" | 0 | |||
| align="center" | 0 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 581.25 | |||
| align="center" | 1250 | |||
|} | |||
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total) | |||
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 | |||
vii. 72C 2 min | |||
viii. 16C hold | |||
4. Pool 3 samples together (300 uL each) | |||
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | |||
6. Measure in nanodrop | |||
===Nanodrop Results=== | |||
{| 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="15" | | |||
|style="font-weight:bold" width="65" | V4S1A | |||
|style="font-weight:bold" width="65" | V4S1B | |||
|style="font-weight:bold" width="65" | V4S1C | |||
|style="font-weight:bold" width="65" | V4S1D | |||
|style="font-weight:bold" width="65" | V6S3A | |||
|style="font-weight:bold" width="65" | V6S3B | |||
|style="font-weight:bold" width="65" | V6S3C | |||
|style="font-weight:bold" width="65" | V6S3D | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 16.2 | |||
| align="center" | 19.9 | |||
| align="center" | 16.1 | |||
| align="center" | 18.9 | |||
| align="center" | 92.2 | |||
| align="center" | 92.9 | |||
| align="center" | 98.2 | |||
| align="center" | 84.5 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | ug in 40 uL | |||
| align="center" | 0.6 | |||
| align="center" | 0.8 | |||
| align="center" | 0.6 | |||
| align="center" | 0.8 | |||
| align="center" | 3.7 | |||
| align="center" | 3.7 | |||
| align="center" | 3.9 | |||
| align="center" | 3.4 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
|style="font-weight:bold" height="15" | total ug | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 2.8 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 14.7 | |||
|} | |||
==DpnII Digestion== | |||
1. Reaction table | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="15" | Sample | |||
| width="65" | V4S1A | |||
| width="65" | V4S1B | |||
| width="65" | V4S1C | |||
| width="65" | V4S1D | |||
| width="65" | V6S3A | |||
| width="65" | V6S3B | |||
| width="65" | V6S3C | |||
| width="65" | V6S3D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 16.2 | |||
| align="center" | 19.9 | |||
| align="center" | 16.1 | |||
| align="center" | 18.9 | |||
| align="center" | 92.2 | |||
| align="center" | 92.9 | |||
| align="center" | 98.2 | |||
| align="center" | 84.5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 35 uL | |||
| align="center" | 0.6 | |||
| align="center" | 0.7 | |||
| align="center" | 0.6 | |||
| align="center" | 0.7 | |||
| align="center" | 3.2 | |||
| align="center" | 3.3 | |||
| align="center" | 3.4 | |||
| align="center" | 3.0 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="30" | uL 10X buffer | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="30" | uL DpnII (50U/uL) | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL nfH2O | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Total | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
|} | |||
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-10-4|tomorrow]] |
Latest revision as of 00:04, 4 October 2013
FISH (Started 10/01/2013)[edit]
Microscopy[edit]
Used the Olympus Microscope upstairs to view the most recent round of FISH slides.
Results[edit]
Control (BSPP-2)
File:Overlay con 10x FISH2013 10 03.png | File:Overlay con 40x FISH2013 10 03.png |
10x magnification; BF,DAPI,TXRED | 40x magnification; BF,DAPI,TXRED |
Experiment (V6S1+V6S3)
File:Overlay exp 20x FISH 2013 10 03.png |
20x magnification; BF,DAPI,TXRED |
Discussion[edit]
The control sample worked well enough, it seems like there is plenty of signal but it isn't in any particular place. The experiment group, however, looked terrible, as it looked like there were no cells anywhere on the dish. Further, there were scratches everywhere, distorting the view. I'll have to make sure on future samples that the cells, after fixation, look healthy and plentiful.
V6S3/V4S1 Probe Production (Amino primers)[edit]
Since I'm out of V6S3 and V4S1 I'm going to make some more.
qPCR Amplification[edit]
1. Reaction Table
20 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V41U-Amino | 100 uM AP2V4 | 100 uM AP1V61U-Amino | 100 uM AP2V6 | H20 | Total Volume | |
std 100 uL rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 46.5 | 100 |
V4S1 (12.5x) | 2.5 | 125 | 5 | 5 | 0 | 0 | 581.25 | 1250 |
V6S3 (12.5x) | 2.5 | 125 | 0 | 0 | 5 | 5 | 581.25 | 1250 |
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total) 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 vii. 72C 2 min viii. 16C hold 4. Pool 3 samples together (300 uL each) 5. Column Purification, elute with 40 uL EB 6. Measure in nanodrop
Nanodrop Results[edit]
V4S1A | V4S1B | V4S1C | V4S1D | V6S3A | V6S3B | V6S3C | V6S3D | |
ng/uL | 16.2 | 19.9 | 16.1 | 18.9 | 92.2 | 92.9 | 98.2 | 84.5 |
ug in 40 uL | 0.6 | 0.8 | 0.6 | 0.8 | 3.7 | 3.7 | 3.9 | 3.4 |
total ug | 2.8 | 14.7 |
DpnII Digestion[edit]
1. Reaction table
Sample | V4S1A | V4S1B | V4S1C | V4S1D | V6S3A | V6S3B | V6S3C | V6S3D |
ng/uL | 16.2 | 19.9 | 16.1 | 18.9 | 92.2 | 92.9 | 98.2 | 84.5 |
ug in 35 uL | 0.6 | 0.7 | 0.6 | 0.7 | 3.2 | 3.3 | 3.4 | 3.0 |
uL 10X buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL DpnII (50U/uL) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
uL nfH2O | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
Total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued tomorrow