Alu Control[edit]
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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]
File:2014-01-08-Alu1.png
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
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: 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]
File:2014-01-08Alu2.png
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.