Kun:LabNotes/ExonomeSeq/2009-5-23: Difference between revisions
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===PCR=== | ===PCR=== | ||
x2 | x2 | ||
DNA 10ul | |||
Solexa_PCR_up(10uM) 2ul | |||
Solexa_PCR_lo(10uM) 2ul | |||
2x iProof master mix 50ul | |||
50x SYBGI 0.4ul | |||
ddH2O 36ul | |||
98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) | |||
5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold. | |||
==Added another round in parallel== | |||
===PCR with dUTP=== | |||
x 2 x 4 | |||
Template: 1.5ul | |||
2x EconoTaq Master Mix 50ul 400 | |||
100uM AmpF6.3NH2 0.2ul 1.6 | |||
100uM AmpR6.3NH2 0.2ul 1.6 | |||
10mM dUTP 0.6ul 4.8 | |||
50x SYBG I 0.4ul 3.2 | |||
H2O 48ul 350 | |||
94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C | |||
Purified every 200ul for each amplicon with one Qiaquick column. | |||
A: 54ng/ul x 28ul | |||
B: 45ng/ul x 28ul | |||
C: 40ng/ul x 28ul | |||
D: 38ng/ul x 28ul | |||
===USER digestion=== | |||
Qiaquick purified DNA 18ul | |||
USER enzyme 2ul | |||
37C 1h | |||
===S1 nuclease digestion=== | |||
10 x S1 nuclease buffer: 3ul | |||
DNA after USER digestion: 20ul | |||
S1 nuclease (10U/ul): 2ul | |||
ddH2O 4ul | |||
37C 10mins. | |||
Purified with MinElute cloumn. Elute in 18ul H2O. | |||
A: 12ng/ul | |||
B: 7.4ng/ul | |||
C: 7.5ng/ul | |||
D: 12ng/ul | |||
===End repair (Enzymatics End-repair Mix)=== | |||
Fragmented DNA 15ul | |||
dNTP 2.5ul | |||
10x buffer 2.5ul | |||
Enzyme mix 1ul | |||
H2O 4ul | |||
Incubate at RT for 30 minutes | |||
Purified with Qiaquick column. | |||
Select the ~100bp fragments with 2% SizeSelect gel | |||
===Adaptor ligation(Enzymatic T4 ligase)=== | |||
NEB | |||
DNA 34ul | |||
2X Rapid Ligation buffer 40ul | |||
50uM Solexa_1 adaptor 2ul | |||
50uM Solexa_2 adaptor 2ul | |||
T4 DNA ligase(600U/ul) 2ul | |||
Keep at room temperature (~25C) for 15 minutes. Purify with MinElute columns. | |||
===Nick translation=== | |||
DNA 8ul | |||
10x ThermoPol buffer 1ul | |||
10mM dNTP 0.4ul | |||
Bst Pol (8U/ul) 0.5ul | |||
65C 10min | |||
===PCR=== | |||
DNA 10ul | DNA 10ul | ||
Solexa_PCR_up(10uM) 2ul | Solexa_PCR_up(10uM) 2ul |
Revision as of 22:40, 23 May 2009
Shotgun library construction
- Start with four normalized and size-selected libraries captured from PGP1-3, NA12878 (prepared by Alice Li). The estimated concentration is ~2ng/ul each.
PCR with dUTP
x 4 x 4 Template: 1ul 2x EconoTaq Master Mix 50ul 800 100uM AmpF6.3NH2 0.2ul 3.2 100uM AmpR6.3NH2 0.2ul 3.2 10mM dUTP 0.6ul 9.6 50x SYBG I 0.4ul 6.4 H2O 48ul 770 94C 3min -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified every 4x50ul for each amplicon with two MinElute column. A: 23ng/ul x 20ul B: 14ng/ul x 20ul C: 14ng/ul x 20ul D: 18ng/ul x 20ul
Purified each of the remaining amplicons with one DNA concentrator-5 column, eluted with 20ul EB A: 6.3ng/ul x 20ul B: 6.2ng/ul x 20ul C: 6.8ng/ul x 20ul D: 4.8ng/ul x 20ul
USER digestion
Qiaquick purified DNA 20ul USER enzyme 2ul 37C 1h DNA concentrator purified DNA 18ul USER enzyme 1ul 37C 1h
S1 nuclease digestion
10 x S1 nuclease buffer: 3ul DNA after USER digestion: 22ul S1 nuclease (10U/ul): 2ul ddH2O 2ul
37C 15mins. Combined two tubes for each library. Purified with MinElute cloumn. Elute in 18ul H2O.
A: 12ng/ul B: 7.4ng/ul C: 7.5ng/ul D: 12ng/ul
End repair (Enzymatics End-repair Mix)
Fragmented DNA 15ul dNTP 2.5ul 10x buffer 2.5ul Enzyme mix 1ul H2O 4ul Incubate at RT for 30 minutes Purified with Qiaquick column. Select the ~100bp fragments with 2% SizeSelect gel
Adaptor ligation(Enzymatic T4 ligase)
NEB DNA 20ul 2X Rapid Ligation buffer 25ul 100uM Solexa_1 adaptor 0.5ul 100uM Solexa_2 adaptor 0.5ul T4 DNA ligase(600U/ul) 1ul H2O 3ul
Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns.
Nick translation
DNA 8ul 10x ThermoPol buffer 1ul 10mM dNTP 0.4ul Bst Pol (8U/ul) 0.5ul 65C 10min
PCR
x2 DNA 10ul Solexa_PCR_up(10uM) 2ul Solexa_PCR_lo(10uM) 2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 36ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) 5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
Added another round in parallel
PCR with dUTP
x 2 x 4 Template: 1.5ul 2x EconoTaq Master Mix 50ul 400 100uM AmpF6.3NH2 0.2ul 1.6 100uM AmpR6.3NH2 0.2ul 1.6 10mM dUTP 0.6ul 4.8 50x SYBG I 0.4ul 3.2 H2O 48ul 350 94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified every 200ul for each amplicon with one Qiaquick column. A: 54ng/ul x 28ul B: 45ng/ul x 28ul C: 40ng/ul x 28ul D: 38ng/ul x 28ul
USER digestion
Qiaquick purified DNA 18ul USER enzyme 2ul 37C 1h
S1 nuclease digestion
10 x S1 nuclease buffer: 3ul DNA after USER digestion: 20ul S1 nuclease (10U/ul): 2ul ddH2O 4ul
37C 10mins. Purified with MinElute cloumn. Elute in 18ul H2O.
A: 12ng/ul B: 7.4ng/ul C: 7.5ng/ul D: 12ng/ul
End repair (Enzymatics End-repair Mix)
Fragmented DNA 15ul dNTP 2.5ul 10x buffer 2.5ul Enzyme mix 1ul H2O 4ul Incubate at RT for 30 minutes Purified with Qiaquick column. Select the ~100bp fragments with 2% SizeSelect gel
Adaptor ligation(Enzymatic T4 ligase)
NEB DNA 34ul 2X Rapid Ligation buffer 40ul 50uM Solexa_1 adaptor 2ul 50uM Solexa_2 adaptor 2ul T4 DNA ligase(600U/ul) 2ul
Keep at room temperature (~25C) for 15 minutes. Purify with MinElute columns.
Nick translation
DNA 8ul 10x ThermoPol buffer 1ul 10mM dNTP 0.4ul Bst Pol (8U/ul) 0.5ul 65C 10min
PCR
DNA 10ul Solexa_PCR_up(10uM) 2ul Solexa_PCR_lo(10uM) 2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 36ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) 5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.