Daniel:Notebook/HiResChrPaint/2013-7-22: Difference between revisions
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|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="30" | | | height="30" | V4S2 (12.5x) | ||
| align="center" | 2.5 | | align="center" | 2.5 | ||
| align="center" | 625 | | align="center" | 625 | ||
Line 47: | Line 47: | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="30" | | | height="30" | V6S2 (12.5x) | ||
| align="center" | 2.5 | | align="center" | 2.5 | ||
| align="center" | 625 | | align="center" | 625 | ||
Line 72: | Line 72: | ||
4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) | 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) | ||
5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue | 5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue | ||
6. Incubated | 6. Incubated 2 hours; resuspend in 50 uL nfH20** | ||
7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | 7. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB | ||
8. Measure concentrations in nanodrop | 8. Measure concentrations in nanodrop | ||
Note(**): It would seem that the time for ethanol precipitation might actually be an important step. Comparing this trial to previous S2 amplifications, incubating [[Daniel:Notebook/HiResChrPaint/2013-6-1|overnight]], the overnight incubation had much higher yield. So it might do to rethink my pipeline. | |||
===qPCR Results=== | ===qPCR Results=== | ||
Line 81: | Line 84: | ||
===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" | V4S4A | |||
| width="65" | V4S4B | |||
| width="65" | V4S4C | |||
| width="65" | V4S4D | |||
| width="65" | V6S4A | |||
| width="65" | V6S4B | |||
| width="65" | V6S4C | |||
| width="65" | V6S4D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 52.6 | |||
| align="center" | 44.5 | |||
| align="center" | 52.1 | |||
| align="center" | 47.4 | |||
| align="center" | 105.7 | |||
| align="center" | 111.3 | |||
| align="center" | 98.1 | |||
| align="center" | 103.3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 40 uL | |||
| align="center" | 2.1 | |||
| align="center" | 1.8 | |||
| align="center" | 2.1 | |||
| align="center" | 1.9 | |||
| align="center" | 4.2 | |||
| align="center" | 4.5 | |||
| align="center" | 3.9 | |||
| align="center" | 4.1 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | Total ug | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 5.8 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | 12.5 | |||
|} | |||
==DpnII Digestion== | |||
1. Prepare Samples | |||
{| 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" | V4S4A | |||
| width="65" | V4S4B | |||
| width="65" | V4S4C | |||
| width="65" | V4S4D | |||
| width="65" | V6S4A | |||
| width="65" | V6S4B | |||
| width="65" | V6S4C | |||
| width="65" | V6S4D | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 52.6 | |||
| align="center" | 44.5 | |||
| align="center" | 52.1 | |||
| align="center" | 47.4 | |||
| align="center" | 105.7 | |||
| align="center" | 111.3 | |||
| align="center" | 98.1 | |||
| align="center" | 103.3 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | ug in 35 uL | |||
| align="center" | 2.1 | |||
| align="center" | 1.8 | |||
| align="center" | 2.1 | |||
| align="center" | 1.9 | |||
| align="center" | 4.2 | |||
| align="center" | 4.5 | |||
| align="center" | 3.9 | |||
| align="center" | 4.1 | |||
|- 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; [[Daniel:Notebook/HiResChrPaint/2013-7-23|Continued 7/23/2013]] |
Latest revision as of 22:30, 23 July 2013
S2 Probe Preparation[edit]
Despite mixed reviews from the S4 set, I'm going to go ahead and make the S2 set. Also, I talked with Alan and he and I are going to prep some cells tomorrow. I'll probably go ahead and attempt fish with the S3 set, since that one worked out great. If I have other probes available, I may use them as well, but Alan and I won't be preparing the cells until tomorrow afternoon, which gives me plenty of time to prep a new set.
PCR Amplification[edit]
1. Reaction Table
20 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V41U | 100 uM AP2V4 | 100 uM AP1V61U | 100 uM AP2V6 | 2 mM aa-dUTP | H20 | Total Volume | |
per rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 2.5 | 45.7 | 100 |
V4S2 (12.5x) | 2.5 | 625 | 5 | 5 | 0 | 0 | 31 | 581.5 | 1250 |
V6S2 (12.5x) | 2.5 | 625 | 0 | 0 | 5 | 5 | 31 | 581.5 | 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. Ethanol Precipitation, added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue 6. Incubated 2 hours; resuspend in 50 uL nfH20** 7. Column Purification, elute with 40 uL EB 8. Measure concentrations in nanodrop
Note(**): It would seem that the time for ethanol precipitation might actually be an important step. Comparing this trial to previous S2 amplifications, incubating overnight, the overnight incubation had much higher yield. So it might do to rethink my pipeline.
qPCR Results[edit]
Nanodrop Results[edit]
Sample | V4S4A | V4S4B | V4S4C | V4S4D | V6S4A | V6S4B | V6S4C | V6S4D |
ng/uL | 52.6 | 44.5 | 52.1 | 47.4 | 105.7 | 111.3 | 98.1 | 103.3 |
ug in 40 uL | 2.1 | 1.8 | 2.1 | 1.9 | 4.2 | 4.5 | 3.9 | 4.1 |
Total ug | 5.8 | 12.5 |
DpnII Digestion[edit]
1. Prepare Samples
Sample | V4S4A | V4S4B | V4S4C | V4S4D | V6S4A | V6S4B | V6S4C | V6S4D |
ng/uL | 52.6 | 44.5 | 52.1 | 47.4 | 105.7 | 111.3 | 98.1 | 103.3 |
ug in 35 uL | 2.1 | 1.8 | 2.1 | 1.9 | 4.2 | 4.5 | 3.9 | 4.1 |
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 7/23/2013