Daniel:Notebook/HiResChrPaint/2013-12-18: Difference between revisions

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=ALU Control=
=ALU Control=
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]]


I have the ALU primers in and I'm testing whether or not I can amplify working probes from genomic DNA.
I have the ALU primers in and I'm testing whether or not I can amplify working probes from genomic DNA.
Line 42: Line 44:
Since I seem to have genomic DNA, I can now test the primers by doing a PCR run.
Since I seem to have genomic DNA, I can now test the primers by doing a PCR run.


==qPCR Amplification==
==qPCR Primary Amplification==


This is only a test run, so I'm going to do it in triplicate (if this succeeds I'll have enough DNA to run the DpnII digestion).
This is only a test run, so I'm going to do it in triplicate (if this succeeds I'll have enough DNA to run the DpnII digestion).
Line 53: Line 55:
|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 AP2V6
|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" | H20
|style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume
|style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume
Line 88: Line 90:




  2. Aliquot 100 uL into each of 12 qPCR tubes  
  2. Aliquot 100 uL into each of 3 qPCR tubes  
  3. PCR protocol:
  3. PCR protocol:
     i. PCR 3 min 95C
     i. PCR 3 min 95C
Line 98: Line 100:
     vii. 72C 2 min
     vii. 72C 2 min
     viii. 16C hold
     viii. 16C hold
  4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total)
  4. Pool 3 samples together each
  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
Line 178: Line 180:
  3. Column Purification, elute with 30 uL EB
  3. Column Purification, elute with 30 uL EB
  4. Measure concentrations in nanodrop
  4. Measure concentrations in nanodrop
===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" | PGP1F
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 13.4
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 30 uL
| align="center" | 0.4
|}
===Gel Image===
[[Image:2013-12-18-ALUTest.jpg|600px]]
==qPCR Secondary 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" | &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 AP1V61U-Amino
|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-A (1X)
| 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-B (1X)
| align="center" | 1
| align="center" | 50
| align="center" | 0.5
| align="center" | 0.5
| align="center" | 48
| align="center" | 100
|}
2. 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
3. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 30 uL EB
4. Measure concentrations in nanodrop
===qPCR Results===
[[Image:2013-12-18-ALUTest2.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" | PGP1F-A
| width="65" | PGP1F-B
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 36.5
| align="center" | 27.4
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 30 uL
| align="center" | 1.1
| align="center" | 0.8
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | total
| align="center" | &nbsp;
| align="center" | 1.9
|}

Latest revision as of 22:57, 8 January 2014

ALU Control[edit]

Back to Calendar

I have the ALU primers in and I'm testing whether or not I can amplify working probes from genomic DNA.

Genomic DNA Extraction[edit]

I did this using PGP1F fibroblasts from my cell culture over in CERC.

1. Trypsinize cells using 1 mL Trypsin LE
2. Resuspend in 10 mL DMEM
3. Spin at 1000 rpm for 3 minutes, remove supernatant
4. Resuspend in 1 mL Lysis buffer from Zymo kit
5. Follow Zymo kit instructions for monolayer cells

Nanodrop Results[edit]

I separated the DNA into two columns, so there are two samples (A and B)

Sample PGP1F-A PGP1F-B
ng/uL 37.4 27.4
ug in 100 uL 3.7 2.7
total 6.5

Since I seem to have genomic DNA, I can now test the primers by doing a PCR run.

qPCR Primary Amplification[edit]

This is only a test run, so I'm going to do it in triplicate (if this succeeds I'll have enough DNA to run the DpnII digestion).

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 (3.1X) 3.1 155 1.55 1.55 148.8 310
PGP1F-B (3.1X) 3.1 155 1.55 1.55 148.8 310


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:2013-12-18-ALUTest.png

Nanodrop Results[edit]

Sample PGP1F-A PGP1F-B
ng/uL 12.9 13.4
ug in 40 uL 0.5 0.5
total 1.0

So first round PCR is not at all lucrative. This product will still be used for several tests, however. The first test will be for DpnII cleavage. I'll also check to see if second round PCR works.

DpnII Digestion[edit]

1. Reaction Table
Sample PGP1F-A PGP1F-B
ng/uL 12.9 13.4
ug in 30 uL 0.4 0.4
uL 10X buffer 5 5
uL DpnII (50U/uL) 1 1
uL nfH2O 14 14
Total 50 50
2. Incubate samples at 37C for 2 hours; 20 minute heat shock at 65C
3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop

Nanodrop Results[edit]

Sample PGP1F
ng/uL 13.4
ug in 30 uL 0.4

Gel Image[edit]

File:2013-12-18-ALUTest.jpg

qPCR Secondary Amplification[edit]

1. Reaction Table
  gDNA 2X Kapa SYBR qPCR MM 100 uM AP1V61U-Amino 100 uM AP2V6 H20 Total Volume
per rxn 1 50 0.5 0.5 48 100
PGP1F-A (1X) 1 50 0.5 0.5 48 100
PGP1F-B (1X) 1 50 0.5 0.5 48 100
2. 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
3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop

qPCR Results[edit]

File:2013-12-18-ALUTest2.png

Nanodrop Results[edit]

Sample PGP1F-A PGP1F-B
ng/uL 36.5 27.4
ug in 30 uL 1.1 0.8
total   1.9