Daniel:Notebook/HiResChrPaint/2014-1-8: Difference between revisions

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>Djacobse
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===Nanodrop Results===
===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" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| 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.

Revision as of 17:49, 9 January 2014

Alu Control

Back to Calendar

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

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

File:2014-01-08-Alu1.png

Nanodrop Results

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

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

File:2014-01-08-Alu1.jpg

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: ng/uL.

Production qPCR

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

File:2014-01-08Alu2.png

Nanodrop Results

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.