Revision as of 01:51, 16 November 2013 by >Djacobse
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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