V6S3 and V4S1 Amplification Amino Primers (Started 10/03/2013)[edit]
Back to Calendar
DpnII Digestion[edit]
3. Column purification, elute with 30 uL EB
4. Measure Concentration in nanodrop
5. Check digestion results with gel
Gel Results[edit]
File:20131004 DpnIIDigest.jpg
Nanodrop Results[edit]
|
V4S1
|
V6S3AB
|
V6S3CD
|
ng/uL
|
39.9
|
175.6
|
152
|
ug in 30 uL
|
1.2
|
5.3
|
4.6
|
ug total
|
1.2
|
|
9.8
|
Discussion[edit]
The V4S1 really doesn't have a lot of DNA left, so I'm going to stop here and amplify the V4 set again before I continue on.
V4S1 Amplification[edit]
1. Reaction Table
|
20 nM OligoPool
|
2X Kapa SYBR qPCR MM
|
100 uM AP1V41U-Amino
|
100 uM AP2V4
|
H20
|
Total Volume
|
std 100 uL rxn
|
0.2
|
50
|
0.4
|
0.4
|
49
|
100
|
V4S1 (12.5x)
|
2.5
|
125
|
5
|
5
|
612.5
|
1250
|
2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total)
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. Pool 3 samples together (300 uL each)
5. Column Purification, elute with 40 uL EB
6. Measure in nanodrop
qPCR Results[edit]
File:2013-10-04-V4S1.png
Nanodrop Results[edit]
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
ng/uL
|
76.5
|
76.3
|
76.3
|
68.3
|
ug in 40 uL
|
3.1
|
3.1
|
3.1
|
2.7
|
ug total
|
|
|
|
11.9
|
DpnII Digestion[edit]
1. Reaction table
Sample
|
V4S1A
|
V4S1B
|
V4S1C
|
V4S1D
|
ng/uL
|
76.5
|
76.3
|
76.3
|
68.3
|
ug in 35 uL
|
2.7
|
2.7
|
2.7
|
2.4
|
uL 10X buffer
|
5
|
5
|
5
|
5
|
uL DpnII (50U/uL)
|
2
|
2
|
2
|
2
|
uL nfH2O
|
8
|
8
|
8
|
8
|
Total
|
50
|
50
|
50
|
50
|
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued tomorrow
Split cells today, including making two dishes for FISH.
From the dishes above.
Protocol[edit]
1. Rinse slides briefly in 1X PBS
2. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
3. Rinse briefly with 1 mL 1X PBS
4. Incubate for 5 minutes in 1 mL 2X SSCT
5. Incubate for 5 minutes in 1 mL 2X SSCT + 50% (v/v) formamide
6. Aspirate, and add 1mL of 2X SSCT + 50% (v/v) formamide for storage at 4C
7. Slides are typically best used within 1-2 weeks of creation