Daniel:Notebook/HiResChrPaint/2013-11-15

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Positive Control (Started 11/14/2013)[edit]

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Lambda Exonuclease Digestion[edit]

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[edit]

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

Positive Control 2 (V6S3 & V6S4)[edit]

qPCR Amplification[edit]

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[edit]

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

Nanodrop Results[edit]

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[edit]

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[edit]

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[edit]

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

File:2013-11-15-DpnII.jpg