Revision as of 01:26, 16 February 2013 by >Alan6017518
[1]
Expansion PCR[edit]
- We will set up 8 reactions for each primer-oligo set (8 in total)
|
per reaction
|
V4 Set1
|
V4 Set2
|
V4 Set3
|
V4 Set4
|
20nM Oligo Pool
|
0.2
|
1.6
|
1.6
|
1.6
|
1.6
|
2X Kapa SYBR qPCR MM
|
50
|
400
|
400
|
400
|
400
|
100uM pAP1V41U
|
0.4
|
3.2
|
3.2
|
3.2
|
3.2
|
100uM AP2V4
|
0.4
|
3.2
|
3.2
|
3.2
|
3.2
|
2mM aa-dUTP
|
3
|
24
|
24
|
24
|
24
|
H2O
|
46
|
368
|
368
|
368
|
368
|
Total Volume
|
100
|
800
|
800
|
800
|
800
|
|
|
|
|
|
|
|
per reaction
|
V6 Set1
|
V6 Set2
|
V6 Set3
|
V6 Set4
|
20nM Oligo Pool
|
0.2
|
1.6
|
1.6
|
1.6
|
1.6
|
2X Kapa SYBR qPCR MM
|
50
|
400
|
400
|
400
|
400
|
100uM pAP1V61U
|
0.4
|
3.2
|
3.2
|
3.2
|
3.2
|
100uM AP2V6
|
0.4
|
3.2
|
3.2
|
3.2
|
3.2
|
2mM aa-dUTP
|
3
|
24
|
24
|
24
|
24
|
H2O
|
46
|
368
|
368
|
368
|
368
|
Total Volume
|
100
|
800
|
800
|
800
|
800
|
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
QPCR Results[edit]
EtOH Precipitation[edit]
- Pool all reactions into 15ml tube (900ul in total)
- add 3ul of glycoblue
- add 90ul of 3M NaoAc
- add 2250ul of 100% EtOH
- Freeze the tube in -80C for 1hr
- centrifuge at 3000rpm for 30min, 4C
- remove supernatant and add 600ul of chilled 75% ETOH and transfer the DNA pellet to a 1.5ml tube (5000ul-3750ul100% EtOH + 1250ul ddh2o)
- spin at 14,000rpm for 5min
- dry the pellet in the hood and resuspend w 100ul ddh2o
- re-purify the amplicons with 2 Qiaquick columns, elute w 40ul EB for each column
- measure concentration w nanodrop
Nanodrop Results[edit]
Probe Set
|
ng/ul
|
ul
|
ng
|
V4S1
|
27
|
80
|
2160
|
V4S2
|
47.7
|
80
|
3816
|
V4S3
|
61.6
|
80
|
4928
|
V4S4
|
35.9
|
80
|
2872
|
V6S1
|
61.2
|
80
|
4896
|
V6S2
|
49.7
|
80
|
3976
|
V6S3
|
74.9
|
80
|
5992
|
V6S4
|
86.7
|
80
|
6936
|
DpnII Digestion[edit]
- We will setup a small testing reaction to optimize the restriction endonuclease reaction
- I will test the reaction with 1ug of probes
Probe Set
|
ng/ul
|
To get 1ug
|
10X DpnII Buffer
|
DpnII
|
H2O
|
Total
|
V4S1
|
27
|
37.04
|
5
|
1
|
6.96
|
50
|
V4S2
|
47.7
|
20.96
|
5
|
1
|
23.04
|
50
|
V4S3
|
61.6
|
16.23
|
5
|
1
|
27.77
|
50
|
V4S4
|
35.9
|
27.86
|
5
|
1
|
16.14
|
50
|
V6S1
|
61.2
|
16.34
|
5
|
1
|
27.66
|
50
|
V6S2
|
49.7
|
20.12
|
5
|
1
|
23.88
|
50
|
V6S3
|
74.9
|
13.35
|
5
|
1
|
30.65
|
50
|
V6S4
|
86.7
|
11.53
|
5
|
1
|
32.47
|
50
|
- Incubate reaction at 37C for 1 hour followed by 20min at 65C for heat inactivation
- Column purification, elute with 30ul EB
PAGE Gel[edit]
File:ZhangLab 2 2013-02-04 14hr 19min.jpg
DpnII Digestion for remaining probes[edit]
Probe Set
|
ng/ul
|
Remaining Volume
|
10X DpnII Buffer
|
DpnII
|
H2O
|
Total
|
V4S1
|
27
|
42.96
|
10
|
5
|
42.04
|
100
|
V4S2
|
47.7
|
59.04
|
10
|
6
|
24.96
|
100
|
V4S3
|
61.6
|
63.77
|
10
|
7
|
19.23
|
100
|
V4S4
|
35.9
|
52.14
|
10
|
6
|
31.86
|
100
|
V6S1
|
61.2
|
63.66
|
10
|
7
|
19.34
|
100
|
V6S2
|
49.7
|
59.88
|
10
|
6
|
24.12
|
100
|
V6S3
|
74.9
|
66.65
|
10
|
7
|
16.35
|
100
|
V6S4
|
86.7
|
68.47
|
10
|
7
|
14.53
|
100
|
- Incubate reaction at 37C for 1 hour followed by 20min at 65C for heat inactivation
- Column purification, elute with 30ul EB
- combine the two sets of probes, making the total volume 60ul
Probe Set
|
Concentration (ng/ul)
|
V4S1
|
20.5
|
V4S2
|
40.7
|
V4S3
|
57.7
|
V4S4
|
32.5
|
V6S1
|
53
|
V6S2
|
45
|
V6S3
|
71.2
|
V6S4
|
83.1
|
Lambda exonuclease digestion[edit]
Probe Set
|
Volume
|
10X Lambda Exo Buffer
|
Lambda Exo (10U/ul)
|
H20
|
V4S1
|
60
|
10
|
5
|
25
|
V4S2
|
60
|
10
|
5
|
25
|
V4S3
|
60
|
10
|
5
|
25
|
V4S4
|
60
|
10
|
5
|
25
|
V6S1
|
60
|
10
|
5
|
25
|
V6S2
|
60
|
10
|
5
|
25
|
V6S3
|
60
|
10
|
5
|
25
|
V6S4
|
60
|
10
|
5
|
25
|
- 37C 1hr>75C 10min>Etoh Precipitation
EtOH Precipitation[edit]
- add 1ul of glycoblue
- add 10ul of 3M NaoAc
- add 250ul of 100% EtOH
- Freeze the tube in -80C for 30min
- centrifuge at 10000rpm for 20min, 4C
- no pellet seen after centrifuge, added 1ul extra glycoblue and freeze for 1 hour, will spin at 14000 rpm for 30 min
- doble the volume of all etoh precipitation reagents and spin again, this time we are able to see a blue pellet
- remove supernatant and add 500ul of chilled 75% ETOH and transfer the DNA pellet to a 1.5ml tube (5000ul-3750ul100% EtOH + 1250ul ddh2o)
- spin at 10,000rpm for 5min
- dry the pellet in the hood and resuspend w 100ul ddh2o
TBU Gel quantification[edit]
File:ZhangLab 2 2013-02-14 18hr 12min.jpg
Name
|
Type
|
Volume
|
|
|
|
|
|
|
|
INT*mm2
|
Subtract Background
|
Amount (ng)
|
Dilution
|
Actual Concentration (ng/ul)
|
Actual Amount (ng)
|
U1
|
100BP_1.5ng
|
17252.4105141619
|
8238.9702455405
|
1.5
|
|
|
|
U2
|
200BP_3ng
|
19505.830581319
|
11897.6403545773
|
3
|
|
|
|
U3
|
100BP_0.75ng
|
9267.2702761862
|
3837.1701143566
|
0.75
|
|
|
|
U4
|
200BP_1.5ng
|
12732.8203794676
|
6602.3301967648
|
1.5
|
|
|
|
U5
|
V4S1
|
11703.1303487805
|
3537.3501054213
|
0.572788436370277
|
0.05
|
11.46
|
74.46
|
U6
|
V4S2
|
19698.1905870518
|
9345.380278514
|
2.14931306069451
|
0.05
|
42.99
|
279.41
|
U7
|
V4S3
|
28173.7208396423
|
16057.7104785571
|
3.97129978107829
|
0.05
|
79.43
|
516.27
|
U8
|
V4S4
|
14259.2804249597
|
6477.480193044
|
1.37085376036386
|
0.05
|
27.42
|
178.21
|
U9
|
V6S1
|
23770.1507084057
|
13991.6304169831
|
3.41048552980481
|
0.05
|
68.21
|
443.36
|
U10
|
V6S2
|
18602.5905544004
|
9715.6102895478
|
2.24980784320488
|
0.05
|
45.00
|
292.48
|
U11
|
V6S3
|
31858.4109494546
|
17246.710513992
|
4.29404050010375
|
0.05
|
85.88
|
558.23
|
U12
|
V6S4
|
48582.591447874
|
28414.9908468327
|
7.32554488822642
|
0.05
|
146.51
|
952.32
|