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

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
(15 intermediate revisions by the same user not shown)
Line 5: Line 5:
Following the [[Daniel:Notebook/HiResChrPaint/2013-12-18|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 ([[Daniel:Notebook/HiResChrPaint/2013-12-18|see Genomic DNA Extraction Results]]).
Following the [[Daniel:Notebook/HiResChrPaint/2013-12-18|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 ([[Daniel:Notebook/HiResChrPaint/2013-12-18|see Genomic DNA Extraction Results]]).


=Expansion qPCR=
==Expansion qPCR==


  1. Reaction table
  1. Reaction table
Line 14: Line 14:
|style="background-color:#D9D9D9;font-weight:bold" width="65" | gDNA
|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" | 2X Kapa SYBR qPCR MM
|style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP1V61U-Amino
|style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP1Alu
|style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP2Alu
|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-A (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:2014-01-08-Alu1.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" | ALU1-A
| width="65" | ALU1-B
| width="65" | ALU1-C
| width="65" | ALU1-D
 
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 15.4
| align="center" | 13.5
| align="center" | 15
| align="center" | 15.9
 
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 40 uL
| align="center" | 0.6
| align="center" | 0.5
| align="center" | 0.6
| align="center" | 0.6
 
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | total
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 2.4
 
|}
 
===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" | &nbsp;
|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" | 100 uM AP2V6
|style="background-color:#D9D9D9;font-weight:bold" width="65" | H20
|style="background-color:#D9D9D9;font-weight:bold" width="65" | H20
Line 29: Line 131:


|- style="font-size:12pt" align="center"
|- style="font-size:12pt" align="center"
|style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F-A (12.1X)
|style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F (12.1X)
| align="center" | 12.1
| align="center" | 12.1
| align="center" | 605
| align="center" | 605
Line 52: Line 154:
  5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
  5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
  6. Measure concentrations in nanodrop
  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" | &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.

Latest revision as of 21:23, 9 January 2014

Alu Control[edit]

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[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.