Daniel:Notebook/HiResChrPaint/2014-1-8: Difference between revisions
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===qPCR Gel=== | |||
1. '''For Each Sample''': 1 uL sample, 5 uL TBE, 2 uL dye; load 7.2 uL | |||
{{GelLanes|Lane2=10bp ladder|Lane3=Alu1-A|Lane4=Alu1-B|Lane5=Alu1-C|Lane6=Alu1-D}} | |||
2. Run gel for 30 minutes at 220V | |||
3. Stain 2 minutes with 3 uL SYBR gold | |||
'''Gel Image''' | |||
[[Image:2014-01-08-Alu1.jpg|500px]] | |||
So these appear to all bee the same, and pretty similar to the gel image from [[Daniel:Notebook/HiResChrPaint/2013-12-18|12/18]]. So I'm going to pool the samples and proceed with production qPCR. | |||
Pooled sample concentration: '''17.1 ng/uL'''. | |||
==Production qPCR== | |||
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" | | |||
|style="background-color:#D9D9D9;font-weight:bold" width="65" | gDNA | |||
|style="background-color:#D9D9D9;font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM | |||
|style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP1V6-Am | |||
|style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP2V6 | |||
|style="background-color:#D9D9D9;font-weight:bold" width="65" | H20 | |||
|style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
| height="15" | per rxn | |||
| align="center" | 1 | |||
| align="center" | 50 | |||
| align="center" | 0.5 | |||
| align="center" | 0.5 | |||
| align="center" | 48 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" | |||
|style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F (12.1X) | |||
| align="center" | 12.1 | |||
| align="center" | 605 | |||
| align="center" | 6.05 | |||
| align="center" | 6.05 | |||
| align="center" | 580.8 | |||
| align="center" | 1210 | |||
|} | |||
2. Aliquot 100 uL into each of 3 qPCR tubes | |||
3. PCR protocol: | |||
i. PCR 3 min 95C | |||
ii. 95C 10 sec | |||
iii. 55C 30 sec | |||
iv. 72C 20 sec | |||
v. plate read | |||
vi. goto ii x17 | |||
vii. 72C 2 min | |||
viii. 16C hold | |||
4. Pool 3 samples together each | |||
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | |||
6. Measure concentrations in nanodrop | |||
===qPCR Results=== | |||
[[Image:2014-01-08Alu2.png|500px]] | |||
===Nanodrop Results=== | |||
{| 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" | ALU2-A | |||
| width="65" | ALU2-B | |||
| width="65" | ALU2-C | |||
| width="65" | ALU2-D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 25.1 | |||
| align="center" | 25 | |||
| align="center" | 25 | |||
| align="center" | 22.2 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 40 uL | |||
| align="center" | 1.0 | |||
| align="center" | 1.0 | |||
| align="center" | 1.0 | |||
| align="center" | 0.9 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | total | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 3.9 | |||
|} | |||
So this is smaller than my usual input material. One possibility is the number of cycles. I could only run this one for 17 cycles, which with the probes is more than enough, but from the [[Daniel:Notebook/HiResChrPaint/2013-12-18|12/18 results]] I'd say that the secondary amplification should be run 21 cycles or so. |
Latest revision as of 21:23, 9 January 2014
Alu Control[edit]
Following the results from December 18, it appears that my primers work. However, I used up all the sample from the previous primary amplification, so I'll have to re-amplify from genomic DNA (see Genomic DNA Extraction Results).
Expansion qPCR[edit]
1. Reaction table
gDNA | 2X Kapa SYBR qPCR MM | 100 uM AP1Alu | 100 uM AP2Alu | H20 | Total Volume | |
per rxn | 1 | 50 | 0.5 | 0.5 | 48 | 100 |
PGP1F-A (12.1X) | 12.1 | 605 | 6.05 | 6.05 | 580.8 | 1210 |
2. Aliquot 100 uL into each of 3 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 10 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pool 3 samples together each 5. Column Purification, elute with 40 uL EB 6. Measure concentrations in nanodrop
qPCR Results[edit]
Nanodrop Results[edit]
Sample | ALU1-A | ALU1-B | ALU1-C | ALU1-D |
ng/uL | 15.4 | 13.5 | 15 | 15.9 |
ug in 40 uL | 0.6 | 0.5 | 0.6 | 0.6 |
total | 2.4 |
qPCR Gel[edit]
1. For Each Sample: 1 uL sample, 5 uL TBE, 2 uL dye; load 7.2 uL
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Sample | 10bp ladder | Alu1-A | Alu1-B | Alu1-C | Alu1-D |
2. Run gel for 30 minutes at 220V 3. Stain 2 minutes with 3 uL SYBR gold
Gel Image
So these appear to all bee the same, and pretty similar to the gel image from 12/18. So I'm going to pool the samples and proceed with production qPCR.
Pooled sample concentration: 17.1 ng/uL.
Production qPCR[edit]
1. Reaction Table
gDNA | 2X Kapa SYBR qPCR MM | 100 uM AP1V6-Am | 100 uM AP2V6 | H20 | Total Volume | |
per rxn | 1 | 50 | 0.5 | 0.5 | 48 | 100 |
PGP1F (12.1X) | 12.1 | 605 | 6.05 | 6.05 | 580.8 | 1210 |
2. Aliquot 100 uL into each of 3 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 10 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pool 3 samples together each 5. Column Purification, elute with 40 uL EB 6. Measure concentrations in nanodrop
qPCR Results[edit]
Nanodrop Results[edit]
Sample | ALU2-A | ALU2-B | ALU2-C | ALU2-D |
ng/uL | 25.1 | 25 | 25 | 22.2 |
ug in 40 uL | 1.0 | 1.0 | 1.0 | 0.9 |
total | 3.9 |
So this is smaller than my usual input material. One possibility is the number of cycles. I could only run this one for 17 cycles, which with the probes is more than enough, but from the 12/18 results I'd say that the secondary amplification should be run 21 cycles or so.