Revision as of 21:45, 15 November 2013 by >Djacobse
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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
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