Daniel:Notebook/HiResChrPaint/2013-9-3: Difference between revisions
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=BSPP Control= | =BSPP Control= | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | |||
At Dr. Zhang's recommendation I'm creating negative controls using Dinh/Noi's bisulfite padlock probes (BSPPs). I have obtained the probes from Noi and I have the primers, so I'm going to proceed making probes as I usually do. These probes, however, should not anneal to regular human DNA, as they have been modified for methylation detection. I'm going to be splicing in aminoallyl dUTP and dye-coupling the probes, as usual. I'll also be performing DpnII Digestion and Lambda Exonuclease digestion, as I usually do. | At Dr. Zhang's recommendation I'm creating negative controls using Dinh/Noi's bisulfite padlock probes (BSPPs). I have obtained the probes from Noi and I have the primers, so I'm going to proceed making probes as I usually do. These probes, however, should not anneal to regular human DNA, as they have been modified for methylation detection. I'm going to be splicing in aminoallyl dUTP and dye-coupling the probes, as usual. I'll also be performing DpnII Digestion and Lambda Exonuclease digestion, as I usually do. | ||
'''Samples''' | |||
*BSPP 1-DMR 220K Exp 1 (10nM); 2/8/11 from Noi | |||
*BSPP 2-DMR 220K Exp 2 (10nM); 2/8/11 from Noi | |||
==qPCR Amplification== | ==qPCR Amplification== | ||
Line 12: | Line 19: | ||
| width="65" | 10 nM OligoPool | | width="65" | 10 nM OligoPool | ||
| width="65" | 2X Kapa SYBR qPCR MM | | width="65" | 2X Kapa SYBR qPCR MM | ||
| width="65" | 100 uM AP1V61U | | width="65" | 100 uM AP1V61U | ||
| width="65" | 100 uM AP2V6 | | width="65" | 100 uM AP2V6 | ||
Line 22: | Line 27: | ||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |- style="background-color:#8DB4E2;font-size:12pt" align="center" | ||
| height="15" | per rxn | | height="15" | per rxn | ||
| align="center" | 0. | | align="center" | 0.4 | ||
| align="center" | 50 | | align="center" | 50 | ||
| align="center" | 0.4 | | align="center" | 0.4 | ||
| align="center" | 0.4 | | align="center" | 0.4 | ||
| align="center" | 2.5 | | align="center" | 2.5 | ||
| align="center" | | | align="center" | 46.3 | ||
| align="center" | 100 | | align="center" | 100 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="30" | BSPP 1 (12.5x) | | height="30" | BSPP 1 (12.5x) | ||
| align="center" | | | align="center" | 5 | ||
| align="center" | 625 | | align="center" | 625 | ||
| align="center" | 5 | | align="center" | 5 | ||
| align="center" | 5 | | align="center" | 5 | ||
| align="center" | 31 | | align="center" | 31 | ||
| align="center" | | | align="center" | 579 | ||
| align="center" | 1250 | | align="center" | 1250 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="30" | BSPP 2 (12.5x) | | height="30" | BSPP 2 (12.5x) | ||
| align="center" | | | align="center" | 5 | ||
| align="center" | 625 | | align="center" | 625 | ||
| align="center" | 5 | | align="center" | 5 | ||
| align="center" | 5 | | align="center" | 5 | ||
| align="center" | 31 | | align="center" | 31 | ||
| align="center" | | | align="center" | 579 | ||
| align="center" | 1250 | | align="center" | 1250 | ||
Line 71: | Line 70: | ||
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:2013-09-03-BSPP.png|600px]] | |||
===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" | Probe | |||
| width="65" | BSPP 1A | |||
| width="65" | BSPP 1B | |||
| width="65" | BSPP 1C | |||
| width="65" | BSPP 1D | |||
| width="65" | BSPP 2A | |||
| width="65" | BSPP 2B | |||
| width="65" | BSPP 2C | |||
| width="65" | BSPP 2D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 68.8 | |||
| align="center" | 75.8 | |||
| align="center" | 70.1 | |||
| align="center" | 71.8 | |||
| align="center" | 61.4 | |||
| align="center" | 61.8 | |||
| align="center" | 60.3 | |||
| align="center" | 62.7 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 40 uL | |||
| align="center" | 2.8 | |||
| align="center" | 3.0 | |||
| align="center" | 2.8 | |||
| align="center" | 2.9 | |||
| align="center" | 2.5 | |||
| align="center" | 2.5 | |||
| align="center" | 2.4 | |||
| align="center" | 2.5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Total | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 11.5 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 9.8 | |||
|} | |||
==DpnII Digestion== | |||
1. Reaction Table | |||
{| 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" | BSPP 1A | |||
| width="65" | BSPP 1B | |||
| width="65" | BSPP 1C | |||
| width="65" | BSPP 1D | |||
| width="65" | BSPP 2A | |||
| width="65" | BSPP 2B | |||
| width="65" | BSPP 2C | |||
| width="65" | BSPP 2D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 68.8 | |||
| align="center" | 75.8 | |||
| align="center" | 70.1 | |||
| align="center" | 71.8 | |||
| align="center" | 61.4 | |||
| align="center" | 61.8 | |||
| align="center" | 60.3 | |||
| align="center" | 62.7 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 35 uL | |||
| align="center" | 2.4 | |||
| align="center" | 2.7 | |||
| align="center" | 2.5 | |||
| align="center" | 2.5 | |||
| align="center" | 2.1 | |||
| align="center" | 2.2 | |||
| align="center" | 2.1 | |||
| align="center" | 2.2 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="30" | uL 10X buffer | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
| align="center" | 5 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="30" | uL DpnII (50U/uL) | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
| align="center" | 2 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | uL nfH2O | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
| align="center" | 8 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Total | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
| align="center" | 50 | |||
|} | |||
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-9-4|09/04/2013]] |
Latest revision as of 00:04, 4 October 2013
BSPP Control[edit]
At Dr. Zhang's recommendation I'm creating negative controls using Dinh/Noi's bisulfite padlock probes (BSPPs). I have obtained the probes from Noi and I have the primers, so I'm going to proceed making probes as I usually do. These probes, however, should not anneal to regular human DNA, as they have been modified for methylation detection. I'm going to be splicing in aminoallyl dUTP and dye-coupling the probes, as usual. I'll also be performing DpnII Digestion and Lambda Exonuclease digestion, as I usually do.
Samples
- BSPP 1-DMR 220K Exp 1 (10nM); 2/8/11 from Noi
- BSPP 2-DMR 220K Exp 2 (10nM); 2/8/11 from Noi
qPCR Amplification[edit]
1. Reaction Table
10 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V61U | 100 uM AP2V6 | 2 mM aa-dUTP | H20 | Total Volume | |
per rxn | 0.4 | 50 | 0.4 | 0.4 | 2.5 | 46.3 | 100 |
BSPP 1 (12.5x) | 5 | 625 | 5 | 5 | 31 | 579 | 1250 |
BSPP 2 (12.5x) | 5 | 625 | 5 | 5 | 31 | 579 | 1250 |
2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. Column Purification, elute with 40 uL EB 6. Measure concentrations in nanodrop
qPCR Results[edit]
Nanodrop Results[edit]
Probe | BSPP 1A | BSPP 1B | BSPP 1C | BSPP 1D | BSPP 2A | BSPP 2B | BSPP 2C | BSPP 2D |
ng/uL | 68.8 | 75.8 | 70.1 | 71.8 | 61.4 | 61.8 | 60.3 | 62.7 |
ug in 40 uL | 2.8 | 3.0 | 2.8 | 2.9 | 2.5 | 2.5 | 2.4 | 2.5 |
Total | 11.5 | 9.8 |
DpnII Digestion[edit]
1. Reaction Table
Sample | BSPP 1A | BSPP 1B | BSPP 1C | BSPP 1D | BSPP 2A | BSPP 2B | BSPP 2C | BSPP 2D |
ng/uL | 68.8 | 75.8 | 70.1 | 71.8 | 61.4 | 61.8 | 60.3 | 62.7 |
ug in 35 uL | 2.4 | 2.7 | 2.5 | 2.5 | 2.1 | 2.2 | 2.1 | 2.2 |
uL 10X buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL DpnII (50U/uL) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
uL nfH2O | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
Total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued 09/04/2013