Daniel:Notebook/HiResChrPaint/2013-11-15: Difference between revisions

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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" | V6S3AB
| width="65" | V6S3CD
| width="65" | V6S4AB
| width="65" | V6S4CD
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 169.9
| align="center" | 180
| align="center" | 169.4
| align="center" | 180.1
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 30 uL
| align="center" | 5.1
| align="center" | 5.4
| align="center" | 5.1
| align="center" | 5.4
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | total
| align="center" |
| align="center" | 10.5
| align="center" |
| align="center" | 10.5
|}


===TBE Gel===
===TBE Gel===

Revision as of 01:35, 16 November 2013

Positive Control (Started 11/14/2013)

Back to Calendar

Lambda Exonuclease Digestion

1. Reaction table
Sample V6S1AB V6S1CD V6S2AB V6S2CD
ng/uL 128.6 125.8 147.2 150
ug in 25 uL 3.2 3.1 3.7 3.8
uL added 25 25 25 25
uL 10x buffer 5 5 5 5
uL Lambda Exonuclease (5U/uL) 3 3 3 3
uL nfH2O 17 17 17 17
total 50 50 50 50
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C
3. Pool samples
4. ssDNA column purification, elute with 20 uL nfH2O
5. Measure Concentration in Nanodrop

ssNanodrop Results

Sample ng/uL ug in 20 uL
V6S1 103.7 2.1
V6S2 226.9 4.5

Positive Control 2 (V6S3 & V6S4)

qPCR Amplification

1. Reaction table
  10 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V61U-Amino 100 uM AP2V6 H20 Total Volume
per rxn 0.4 50 0.4 0.4 48.8 100
V6S3 (12.5x) 5 625 5 5 610 1250
V6S4 (12.5x) 5 625 5 5 610 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

File:2013-11-15-V6S3-V6S4.png

Nanodrop Results

Probe V6S3A V6S3B V6S3C V6S3D V6S4A V6S4B V6S4C V6S4D
ng/uL 108.7 107.4 108.1 103.8 113.7 111.4 110.4 113.3
ug in 40 uL 4.3 4.3 4.3 4.2 4.5 4.5 4.4 4.5
Total       17.1       18.0

DpnII Digestion

1. Reaction Table
Sample V6S3A V6S3B V6S3C V6S3D V6S4A V6S4B V6S4C V6S4D
ng/uL 108.7 107.4 108.1 103.8 113.7 111.4 110.4 113.3
ug in 35 uL 3.8 3.8 3.8 3.6 4.0 3.9 3.9 4.0
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 2 hours; 20 minute heat shock at 65C
3. Column Purification, elute with 30 uL EB
4. Measure concentrations in nanodrop

Nanodrop Results

Sample V6S3AB V6S3CD V6S4AB V6S4CD
ng/uL 169.9 180 169.4 180.1
ug in 30 uL 5.1 5.4 5.1 5.4
total 10.5 10.5

TBE Gel

1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7.2 uL for all)


Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 10bp ladder V6S3AB V6S3CD V6S4AB V6S4CD
2. Run for 25 minutes at 220V
3. Stain with 3 uL SYBR gold for 2 minutes

Gel Image