Daniel:Notebook/HiResChrPaint/2013-7-16: Difference between revisions

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  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 2 hours; resuspend in 50 uL nfH20
  6. Incubated 4 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
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===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" | V4S1A
| width="65" | V4S1B
| width="65" | V4S1C
| width="65" | V4S1D
| width="65" | V6S1A
| width="65" | V6S1B
| width="65" | V6S1C
| width="65" | V6S1D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 97.6
| align="center" | 91.6
| align="center" | 99
| align="center" | 99.8
| align="center" | 118.6
| align="center" | 113.8
| align="center" | 117.7
| align="center" | 118.8
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 40 uL
| align="center" | 3.9
| align="center" | 3.7
| align="center" | 4.0
| align="center" | 4.0
| align="center" | 4.7
| align="center" | 4.6
| align="center" | 4.7
| align="center" | 4.8
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total ug
| align="center" |
| align="center" |
| align="center" |
| align="center" | 15.5
| align="center" |
| align="center" |
| align="center" |
| align="center" | 18.8
|}
==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" | V4S3A
| width="65" | V4S3B
| width="65" | V4S3C
| width="65" | V4S3D
| width="65" | V6S3A
| width="65" | V6S3B
| width="65" | V6S3C
| width="65" | V6S3D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 97.6
| align="center" | 91.6
| align="center" | 99
| align="center" | 99.8
| align="center" | 118.6
| align="center" | 113.8
| align="center" | 117.7
| align="center" | 118.8
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 35 uL
| align="center" | 3.4
| align="center" | 3.2
| align="center" | 3.5
| align="center" | 3.5
| align="center" | 4.2
| align="center" | 4.0
| align="center" | 4.1
| align="center" | 4.2
|- 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; [[Daniel:Notebook/HiResChrPaint/2013-7-17|Continued]] 7/17/2013]]

Latest revision as of 18:35, 17 July 2013

S3 Probe Preparation[edit]

Back to Calendar

Since I don't have any S3 set left, I'm going to make a new run using the various new techniques, specifically I am more confident in the dye coupling stage now. I'm going to make just the aa-dUTP (ARES labeling) style for now, since it is difficult to balance the reactions when I have a lot of samples.

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
V4S3 (12.5x) 2.5 625 5 5 0 0 31 581.5 1250
V6S3 (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
    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 4 hours; resuspend in 50 uL nfH20
7. Column Purification, elute with 40 uL EB
8. Measure concentrations in nanodrop

qPCR Results[edit]

File:V4S3-071613.png

Nanodrop Results[edit]

Sample V4S1A V4S1B V4S1C V4S1D V6S1A V6S1B V6S1C V6S1D
ng/uL 97.6 91.6 99 99.8 118.6 113.8 117.7 118.8
ug in 40 uL 3.9 3.7 4.0 4.0 4.7 4.6 4.7 4.8
Total ug 15.5 18.8

DpnII Digestion[edit]

1. Prepare Samples
Sample V4S3A V4S3B V4S3C V4S3D V6S3A V6S3B V6S3C V6S3D
ng/uL 97.6 91.6 99 99.8 118.6 113.8 117.7 118.8
ug in 35 uL 3.4 3.2 3.5 3.5 4.2 4.0 4.1 4.2
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 7/17/2013]]