Revision as of 19:00, 18 May 2014 by >Noi
BSPP capture of amplified DNA from Illumina
Sample list:
- 13 samples received on 2014-03-18 [[1]]. Note: I processed only A1-A10 samples which are amplified DNA samples.
- 8 samples received on 2014-04-14[[2]]
2014-04-07
BSPP capture round1 using Polymerase from Illumina
- 2014-05-05: Received ~30ul of polymerase with property similar to Amplitaq Stoffel fragment. Conc. 3U/ul. She is not allowed to provide any further info. of this enzyme.
- I performed BSPP capture on samples A1-A10 and sample #1-#8
Tube ID
|
Conc. (ng/ul)
|
Volume for 200/300ng (ul)
|
Tube ID
|
H2O (ul)
|
GP1V4 probe
|
10X AmpLigase Buffer
|
Total (ul)
|
Total amount (ng)
|
A1 |
189.77 |
1.58 |
A1 |
14.16 |
2.26 |
2.00 |
20.00 |
300.00
|
A2 |
192.53 |
1.56 |
A2 |
14.18 |
2.26 |
2.00 |
20.00 |
300.00
|
A3 |
142.63 |
2.10 |
A3 |
13.64 |
2.26 |
2.00 |
20.00 |
300.00
|
A4 |
145.67 |
2.06 |
A4 |
13.68 |
2.26 |
2.00 |
20.00 |
300.00
|
A5 |
182.27 |
1.65 |
A5 |
14.09 |
2.26 |
2.00 |
20.00 |
300.00
|
A6 |
164.77 |
1.82 |
A6 |
13.92 |
2.26 |
2.00 |
20.00 |
300.00
|
A7 |
112.93 |
2.66 |
A7 |
13.08 |
2.26 |
2.00 |
20.00 |
300.00
|
A8 |
119.33 |
2.51 |
A8 |
13.23 |
2.26 |
2.00 |
20.00 |
300.00
|
A9 |
188.90 |
1.59 |
A9 |
14.15 |
2.26 |
2.00 |
20.00 |
300.00
|
A10 |
205.10 |
1.46 |
A10 |
14.28 |
2.26 |
2.00 |
20.00 |
300.00
|
NTC |
0.00 |
0.00 |
NTC |
15.74 |
2.26 |
2.00 |
20.00 |
0.00
|
#1 |
34.6 |
5.78 |
#1 |
9.96 |
2.26 |
2.00 |
20.00 |
200.00
|
#2 |
30.4 |
6.58 |
#2 |
9.16 |
2.26 |
2.00 |
20.00 |
200.00
|
#3 |
39.9 |
5.01 |
#3 |
10.73 |
2.26 |
2.00 |
20.00 |
200.00
|
#4 |
41.4 |
4.83 |
#4 |
10.91 |
2.26 |
2.00 |
20.00 |
200.00
|
#5 |
21.6 |
9.26 |
#5 |
6.48 |
2.26 |
2.00 |
20.00 |
200.00
|
#6 |
30.9 |
6.47 |
#6 |
9.27 |
2.26 |
2.00 |
20.00 |
200.00
|
#7 |
36.1 |
5.54 |
#7 |
10.20 |
2.26 |
2.00 |
20.00 |
200.00
|
#8 |
34.4 |
5.81 |
#8 |
9.93 |
2.26 |
2.00 |
20.00 |
200.00
|
NTC |
0 |
0.00 |
NTC |
15.74 |
2.26 |
2.00 |
20.00 |
0.00
|
GP1V4 reaction mix
Components
|
Volume (ul)
|
22 rxn mix
|
10X AmpLigase Buffer |
2.00 |
44.00
|
GP1_V4 (5.75ng/ul) |
2.26 |
49.74
|
Toal |
4.26 |
|
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h
-> add 3ul SLN mix (1.5U/ul Illumina's polymerase (3U/reaction); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN Mix
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume (ul), total 48ul
|
Illumina's Polymerase |
3 |
U/ul |
1.5 |
U/ul |
24.00
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
4.80
|
dNTP |
1 |
mM |
100 |
uM |
4.80
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
4.80
|
H2O |
|
|
|
|
9.60
|
Amplification
Components
|
1x rxn
|
22 rxn mix
|
Captured template |
15.00 |
0.00
|
10uM AmpF6.4Sol |
2.00 |
44.00
|
10uM AmpR6.3.IndX (X=1-16) |
2.00 |
0.00
|
2X KAPA SYBR MM |
50.00 |
1100.00
|
H2O |
31.00 |
682.00
|
Total |
100.00 |
|
- - Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template
- - Use the same PCR profile for library-free protocol
- - Stopped reaction at 21 cycles
Samples
|
Index
|
Samples
|
Index
|
Samples
|
Index
|
A1 |
Ind_41 |
A9 |
Ind_49 |
#1 |
Ind_57
|
A2 |
Ind_42 |
A10 |
Ind_50 |
#2 |
Ind_58
|
A3 |
Ind_43 |
NTC-1 |
Ind_51 |
#3 |
Ind_59
|
A4 |
Ind_44 |
NTC-2 |
Ind_52 |
#4 |
Ind_60
|
A5 |
Ind_45 |
NTC-PCR |
Ind_53 |
#5 |
Ind_61
|
A6 |
Ind_46 |
|
|
#6 |
Ind_62
|
A7 |
Ind_47 |
|
|
#7 |
Ind_63
|
A8 |
Ind_48 |
|
|
#8 |
Ind_64
|
- Verified PCR product in 6% TBE gel by loading 4ul of PCR product
File:ZhangLab 2 2014-05-09 15hr 25min ILMN GP1V4 gel1 A1-A10 NTC.jpg
File:ZhangLab 2 2014-05-09 15hr 27min ILMN GP1V4 gel2 no1-8 NTC.jpg
- Perform AMPure bead purification (0.7X volume)
- Elute with 55ul EB buffer
- Verify and quantify DNA concentration in 6% TBE gel by loading 4ul of bead-purified DNA
File:ZhangLab 2 2014-05-17 17hr 52min PQ4ul A1-A10 Exp5-07.jpg
File:ZhangLab 2 2014-05-17 17hr 52min PQ4ul no1-no8 NTC Exp5-07.jpg