Alice:LabNotes/2009-6-4: Difference between revisions
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==Shotgun library construction== | ==single-end Shotgun sequencing library construction== | ||
Line 10: | Line 10: | ||
1mM dUTP 4ul 16ul | 1mM dUTP 4ul 16ul | ||
50x SYBG I 0.4ul 1.6ul | 50x SYBG I 0.4ul 1.6ul | ||
H2O 50ul | H2O 50ul 200ul | ||
94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C | 94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C | ||
Purified the amplicon with one Qiaquick column each | Purified the amplicon with one Qiaquick column each | ||
Line 18: | Line 18: | ||
f-1: 13.3ng/ul x 30ul | f-1: 13.3ng/ul x 30ul | ||
f-2: 33.6ng/ul x 30ul | f-2: 33.6ng/ul x 30ul | ||
[[Image:ZhangLab_2 2009-06-05 11hr 19min.jpg]] | |||
===USER digestion=== | ===USER digestion=== | ||
Line 32: | Line 34: | ||
37C 15mins | 37C 15mins | ||
Purified with MinElute column. Elute in 18ul H2O. | Purified with MinElute column. Elute in 18ul H2O. | ||
Nanodrop: | |||
e-1:5ng/ul e-2:7.1ng/ul | |||
f-1:6.8ng/ul f-2:10.1ng/ul | |||
===End repair (epicentre)=== | ===End repair (epicentre)=== | ||
pos. control | |||
Fragmented DNA 15ul | Fragmented DNA 15ul Spacer 107bp 2ul + H2O 13ul | ||
dNTP 2.5ul | dNTP 2.5ul | ||
10x buffer 2.5ul | 10x buffer 2.5ul | ||
Line 42: | Line 48: | ||
Incubate at RT for 40 minutes | Incubate at RT for 40 minutes | ||
Purified with minelute column and elute in 20ul | Purified with minelute column and elute in 20ul | ||
Select the ~100bp fragments with Egel. | Select the ~100bp fragments with Egel (only the seq. library samples, positive control not included). | ||
===Adaptor ligation=== | ===Adaptor ligation=== | ||
pos. control neg control | |||
DNA 20ul | DNA 16ul 20ul H2O 16ul | ||
2X Rapid Ligation buffer | 2X Rapid Ligation buffer 18ul 22ul 18ul | ||
100uM Solexa_1 adaptor | 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul | ||
100uM Solexa_2 adaptor | 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul | ||
QuickLigase(400U/ul) | QuickLigase(400U/ul) 1ul 1ul 1ul | ||
Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB. | Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB. | ||
performed one round of size selection on positive control tube using 2% Size Select E-gel. The total amount obtained is | performed one round of size selection on positive control tube using 2% Size Select E-gel. The total amount obtained is 16ul. | ||
===Nick translation=== | ===Nick translation=== | ||
DNA 15ul | DNA 15ul | ||
10x ThermoPol buffer 2ul | 10x ThermoPol buffer 2ul | ||
10mM dNTP 0.4ul | 10mM dNTP 0.4ul | ||
BSA 2ul | BSA 2ul | ||
Bst Pol (8U/ul) 1ul | Bst Pol (8U/ul) 1ul | ||
65C 20min | 65C 20min | ||
TBE gel size selection at 150-170bp | |||
===PCR=== | ===PCR=== | ||
x | x 6 | ||
DNA | DNA 10ul | ||
Solexa_PCR_up(100uM) 0.2ul | |||
Solexa_PCR_lo(100uM) 0.2ul | |||
2x iProof master mix 50ul | |||
50x SYBGI 0.4ul | |||
ddH2O 40ul | |||
98C 30sec -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 7 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold. | |||
[[Image:ZhangLab_2 2009-06-05 16hr 02min.jpg]] | |||
x 6 | |||
DNA 10ul | |||
Solexa_PCR_up(100uM) 0.2ul | Solexa_PCR_up(100uM) 0.2ul | ||
Solexa_PCR_lo(100uM) 0.2ul | Solexa_PCR_lo(100uM) 0.2ul | ||
Line 71: | Line 91: | ||
50x SYBGI 0.4ul | 50x SYBGI 0.4ul | ||
ddH2O 40ul | ddH2O 40ul | ||
98C 30sec -> | 98C 30sec -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 6 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold. | ||
[[Image:ZhangLab_2 2009-06-07 19hr 24min.jpg]] | |||
Qiaquick column purification and elute in 40ul of EB | |||
Nanodrop: | |||
e-1: 24.8ng/ul | |||
e-2: 34.4ng/ul | |||
f-1: 30.0ng/ul | |||
f-2: 34.7ng/ul | |||
===result=== | |||
gel quantification result: | |||
e-1: PGP5-1-060809 (7.43ng/ul = 62.5nM) | |||
e-2: PGP5-2-060809 (7.81ng/ul = 65.7nM) | |||
f-1: PGP7-1-060809 (8.98ng/ul = 75.5nM) | |||
f-2: PGP7-2-060809 (9.47ng/ul = 79.7nM) | |||
send out 10ul of each for sequencing (for lane 4-7) | |||
and also 10ul of f-2: PGP7-2-060809 for sequencing (for lane 8) |
Latest revision as of 21:23, 17 June 2009
single-end Shotgun sequencing library construction[edit]
PCR with dUTP[edit]
x 4 x4 Template: 0.25ul 1ul 2x EconoTaq Master Mix 50ul 200ul 100uM AmpF6.3NH2 0.2ul 0.8ul 100uM AmpR6.3NH2 0.2ul 0.8ul 1mM dUTP 4ul 16ul 50x SYBG I 0.4ul 1.6ul H2O 50ul 200ul 94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified the amplicon with one Qiaquick column each e-1: 14.2ng/ul x 30ul e-2: 31.1ng/ul x 30ul f-1: 13.3ng/ul x 30ul f-2: 33.6ng/ul x 30ul
File:ZhangLab 2 2009-06-05 11hr 19min.jpg
USER digestion[edit]
Qiaquick purified DNA 30ul USER enzyme 2ul 37C 1h
S1 nuclease digestion[edit]
10 x S1 nuclease buffer: 4ul DNA after USER digestion: 32ul S1 nuclease (10U/ul): 1ul ddH2O 3ul
37C 15mins Purified with MinElute column. Elute in 18ul H2O.
Nanodrop: e-1:5ng/ul e-2:7.1ng/ul f-1:6.8ng/ul f-2:10.1ng/ul
End repair (epicentre)[edit]
pos. control Fragmented DNA 15ul Spacer 107bp 2ul + H2O 13ul dNTP 2.5ul 10x buffer 2.5ul enzyme 0.5ul ATP 5ul Incubate at RT for 40 minutes Purified with minelute column and elute in 20ul Select the ~100bp fragments with Egel (only the seq. library samples, positive control not included).
Adaptor ligation[edit]
pos. control neg control DNA 16ul 20ul H2O 16ul 2X Rapid Ligation buffer 18ul 22ul 18ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul QuickLigase(400U/ul) 1ul 1ul 1ul Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB.
performed one round of size selection on positive control tube using 2% Size Select E-gel. The total amount obtained is 16ul.
Nick translation[edit]
DNA 15ul 10x ThermoPol buffer 2ul 10mM dNTP 0.4ul BSA 2ul Bst Pol (8U/ul) 1ul 65C 20min
TBE gel size selection at 150-170bp
PCR[edit]
x 6 DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 7 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold.
File:ZhangLab 2 2009-06-05 16hr 02min.jpg
x 6 DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 6 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold. File:ZhangLab 2 2009-06-07 19hr 24min.jpg
Qiaquick column purification and elute in 40ul of EB Nanodrop: e-1: 24.8ng/ul e-2: 34.4ng/ul f-1: 30.0ng/ul f-2: 34.7ng/ul
result[edit]
gel quantification result: e-1: PGP5-1-060809 (7.43ng/ul = 62.5nM) e-2: PGP5-2-060809 (7.81ng/ul = 65.7nM) f-1: PGP7-1-060809 (8.98ng/ul = 75.5nM) f-2: PGP7-2-060809 (9.47ng/ul = 79.7nM)
send out 10ul of each for sequencing (for lane 4-7) and also 10ul of f-2: PGP7-2-060809 for sequencing (for lane 8)