Kun:LabNotes/ASE/2007-11-13
Exp.1 eSNPs capture (cont. on the 2007-11-11 exp.)[edit]
Perform PCR on 5ul capturing reactions (100ul for gDNA).
x 1 x 12 template: 5ul each ABI 10x buffer: 4.5ul 54ul 25mM MgCl2: 3ul 36ul 10mM dNTP: 1ul 12ul 100uM AmpF2Sol: 0.15ul 1.8ul 100uM AmpRSol: 0.15ul 1.8ul 50X SYBG I: 0.4ul 4.8ul iTaq: 0.5ul 6ul H2O: 35.3ul 430ul
94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.
File:2007-11-14-Hues6-eSNP84k-PCR.png
Perform PAGE purification.
File:2007-11-14-Hues6-seqlib.png
Gel quantification: Hues6 gDNA: 3.64ng/ul => 52nM[WRONG] should be 26nM Hues6 cDNA: 1.76ng/ul => 25nM[WRONG] should be 13nM Hues6(no mouse) cDNA: 2.09ng/ul => 30nM[WRONG] should be 15nM
Solexa libraries shipped to Gary Gao:
First batch, monomer: Hues6gs, 20nM (should be 7nM, corrected before sequencing) Hues6cs, 20nM (should be 7nM, corrected before sequencing) Hues6nms, 20nM (should be 7nM, corrected before sequencing) Second batch, dimer: Hues6gl, 52nM Hues6cl, 25nM Hues6nml, 30nM
Hues6gs and Hues6cs was sequenced on 12/05/2007. Here is the summary of the results:
Summary of reads: Library # reads # mapped % mappable Hues6cs 14,513,045 7,194,901 49.6% Hues6gs 11,035,520 5,477,493 49.6%
# of SNPs with (Fa+Fb)>0.9 and N>20 Hues6gs 14,733 Hues6cs 15,193 Hues6gs&cs 14,318
Genes with >1 heterozygous SNPs: Balanced: 45 (both expressed at the similar level) Allele-specific: 44 (both expressed, one allele is stronger than the other) Mono-allelic: 27 (only one allele is expressed) Conflicting results: 61
Exp.2 Probe prep. for the CES22k set on two new libraries[edit]
Emily resynthesized two libraries for the CES22k set.
- CES22k 01783910053 (received Nov. 2007, same sequences as the above library)
- CES22k 10783810008 (received Nov. 2007, contains Solexa amplification adaptors)
I'm resuspending the oligos in 100ul nuclease free H2O for the concentration of 100nM.
PCR (with iTaq)
x1 x16x2 template(100nM) 0.1ul 2x1.6ul 10x ABI buffer 10ul 32ul 25mM MgCl2 6ul 192ul 10mM dNTP 2ul 64ul 100uM pAP1V6U 0.3ul 9.6ul 100uM phosAP2V6 0.3ul 9.6ul 50X SYBG I 0.8ul 25.6ul iTaq 1ul 32ul H2O 80ul 2560ul
94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Ethanol precipitation, resuspend in 135ul dH2O.
Lambda exo digestion: add 15ul 10X lambda exo buffer, 4ul lambda exo, 37C 4h, 75C 15min, ethanol precipitation, resuspend in 100ul dH2O.
Dpn II & USER digestion: Add 13.5ul 10X Dpn II buffer, 5ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 10ul Dpn II, 5ul USER, 37C 8h, 75C 10min.