Kun:LabNotes/ASE/2008-1-2
Exp.1 Capturing cSNPs with the eSNP84k probe set[edit]
Templates:
- NA20431, 347ng/ul, one reaction with the equal molar pool of all five probe sets, 800ng/reaction.
- GM20431 ds-cDNA, 122ng/ul, Lib #1: 1ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
- GC1EP, 114ng/ul, Lib #1: 10ng; Lib #2: 20ng; Lib #3 50ng; Lib #4: 100ng; Lib #5: 400ng
NA20431 | GM20431 | GC1EP | |||||||||
Template | 2.3 | 1(20ng) | 1(20ng) | 1(50ng) | 0.85 | 3.4 | 1(20ng) | 1(20ng) | 1(50ng) | 0.88 | 3.5 |
10x buffer | 1.5 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Lib #1 (384nM) | 2 | 2 | 2 | ||||||||
Lib #2 (580nM) | 1.5 | 1.5 | 1.5 | ||||||||
Lib #3 (331nM) | 2.6 | 2.6 | 2.6 | ||||||||
Lib #4 (500nM) | 2.4 | 2.4 | 2.4 | ||||||||
Lib #5 (460nM) | 1.8 | 1.8 | 1.8 | ||||||||
H2O | 0.9 | 6 | 6.5 | 5.4 | 5.75 | 3.8 | 6 | 6.5 | 5.4 | 5.72 | 3.7 |
94C 3min->60C 40h-> add 1.5ul/1.0ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 1uM dNTP) -> 60C 1h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul of exo mix (Exo I, Exo III, T7 exo)->37c 2 hour -> 94C 5min -> 4C hold.
Exp.2 Probe prep. for the CES22k set on two new libraries[edit]
PCR (with JumpStart Taq)
x1 x16x2 template(100nM) 0.05ul 2x0.8ul 10x ABI buffer 10ul 320ul 25mM MgCl2 6ul 192ul 10mM dNTP 2ul 64ul 100uM pAP1V6U 0.3ul 9.6ul 100uM phosAP2V6 0.3ul 9.6ul JumpStart Taq 1ul 32ul H2O 80ul 2560ul
94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Purify each amplicons with Bioneer 4 columns. Yield:
- CES22k #4: 63ng/ul x 120ul
- CES22k #5: 62ng/ul x 120ul
Lambda exo digestion: to 120ul DNA, add 15ul 10X lambda exo buffer, 8ul lambda exo, 7ul H2O, 37C 4h, 75C 15min, add 10ul USER enzyme, 37C 2h. Purified 110ul of #4 and 40ul of #5 with Bioneer column, very little DNA was recovered. Ethanol precipitation the remaining DNA, resuspended in 40ul (#4) and 125ul(#5) ddH2O.
Dpn II & USER digestion: Add 5ul/15 10X Dpn II buffer, 2ul/6ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 4ul/10ul Dpn II, 2ul/8ul USER, 37C 4h, 75C 10min.
Purified 55ul of #4 and 100ul of #5 with MinElute columns. Again I lost most DNA, only ~80ng was recovered for each sample. I proceed to PAGE purification on the unpurified #5.
Exp.3 Make gDNA-free cDNA from Hues29, Hues30, Hues33[edit]
RNA clean-up[edit]
Hues29 Hues30 Hues33 total RNA 0.76ug/ul x14ul 0.79ng/ul x 14ul 0.8ng/ul x 12ul 10x buffer 3ul 3ul 3ul H2O (DEPC) 17ul 10ul 0ul DNase I(2u/ul) 2ul 2ul 2ul RNase Inhibitor 0.5ul 0.5ul 0.5ul 37C 30min -> 75C 10min -> Cleanup with RNeasy columns
Yield:
First strand synthesis[edit]
Hues29 Hues30 Hues33 RNA 25ul 25ul 25ul 10mM dNTP 3ul 3ul 3ul 50uM Oligo dT 3ul 3ul 3ul 65C 5min -> place on ice for 1min Add 10x RT buffer 6ul 6ul 6ul 25mM MgCl2 12ul 12ul 12ul 0.1M DTT 6ul 6ul 6ul RNase OUT(40U/ul) 2ul 2ul 2ul SS III RT(200U/ul) 3ul 3ul 1ul 50C 50min -> 85C 5min
Second strand synthesis[edit]
Cleanup the first-strand reactions with G-25 columns.
To the ~60ul reactions, add H2O (DEPC) 22ul 10ul NEBuffer 2 10ul 10mM dNTP 3ul DNA Pol I(10U/ul) 4ul RNase H (2U/ul) 1ul Mix well, incubate at 16C for 2h, purified with MinElute columns.
Yield: Hues29: 11ng/ul x 15ul Hues30: 8ng/ul x 15ul Hues33: 8ng/ul x 15ul Not sure why the yield is so low. I lost a lot of RNA in the clean up step.