Alice:LabNotes/2010-2-15
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Extract DNA using DNeasy Blood and Tissue kit[edit]
material[edit]
iPS 29e P21 received from Eggan Lab on 2/12/2010 (cell pellet with unknown cell count) PGP1F received from Jay Lee on 1/27/2010 (cell pellet with 100,000 cells/tube, and one tube is used)
protocol[edit]
a. centrifuge the appropriate number of cells (max 5 million) for 5 min at 1000g. Resuspend the pellet in 200ul PBS, add 20ul proteinase K *put 400ul PBS into iPS cell pellet, break up the cells and aliquot into 2 tubes with 200ul each [RNA-free gDNA: add 4ul RNase A (100mg/ml), mix by vortexing, incubate for 2 min at RT ] b. add 200ul buffer AL (without added ethanol), mix thoroughly by vortexing, and incubate at 56C for 10 min. c. add 200ul 100% ethanol to the sample, and mix thoroughly by vortexing d. pipet the mixture from last step into the DNeasy Mini spin column placed in a 2 ml collection tube. centrifuge at 8000rpm for 1 min, discard the flow-through and collection tube. e. place the DNeasy Mini spin column placed in a new 2 ml collection tube, add 500ul buffer AW1, and centrifuge at 8000rpm for 1 min, discard the flow-through and collection tube. f. place the DNeasy Mini spin column placed in a new 2 ml collection tube, add 500ul buffer AW2, and centrifuge at 14,000rpm for 3 min to dry DNeasy membrane, discard the flow-through and collection tube. h. place the DNeasy Mini spin column in a clean 1.5ml microcentrifuge tube, and pipet 200ul buffer AE on the membrane. Incubate at RT for 1 min, then centrifuge at 8000rpm for 1min i. maximum yeild, repeat last elution step with a new 1.5ml tube
result[edit]
first elution: PGP1F: 95.7ng/ul * 200ul ips 29e P21: 136.6ng/ul * 200ul ips 29e P21: 134.6ng/ul * 200ul
second elution: PGP1F: 5.3ng/ul * 100ul ips 29e P21: 64.2ng/ul * 100ul ips 29e P21: 45ng/ul * 100ul
Continue the preparation of biotin-labeled probe for exome capture[edit]
Construction of Illumina Genomic libraries[edit]
previous preparation steps can be found under labnotes 1-10-2010 for DF and foreskin sets previous preparation steps can be found under labnotes 1-15-2010 for CViB and CViF sets DF and foreskin sets still have purified post-PCR product after ligation CViB and CViF sets stopped at ligation step
adaptor ligation[edit]
Prepare adaptors (need to be done only for the first time): 100uM PE-t: 20ul 100uM PE-b: 20ul 10x stoffel buffer: 10ul H2O: 50ul 94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec.
commonly used adaptors: Blunt-end adaptors: 5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH Solexa_1_up 3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH Solexa_1_lo_nop TA adaptors (for the one adaptor protocol): 5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH PE_t_adapter 3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos t_adaptor_rc_s regular Y adaptor: PE_t_adaptor(top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond PE_b_adaptor(bottom) \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation
Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. adaptor:target molar ratio is 1:10~20 A-tailed DNA 10 ul 20uM Y adaptor 3 ul (4.3ul for CViB) 5X Quick ligase buffer 10 ul Quick Ligase 3 ul H2O 24 ul (22.7ul for CViB)
Incubate at room temperature for 15 minutes purify the product with Agencourt AMpure kit and elute in 40ul ddH2O
PCR[edit]
Ligation products 5ul (8 well for each set, total of 16 well) 100uM Solexa_PCR_up 0.2ul 100uM solexa_PCR-lo 0.2ul 2X phusion HF master mix 50ul H2O 45ul PCR program: 98 °C 30sec -> 9 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. purify the products with Qiaquick kit and elute in 40ul ddH2 CViB: 48.7ng/ul CViF: 77.7ng/ul File:ZhangLab 2 2010-02-16 14hr 08min.jpg
Biotin-labeled probe capture[edit]
Hybrid selection[edit]
Mix the following to 20ul total volume: 1ug ligated DNA with 100ng padlock probes 2.5ug of human Cot-1 DNA 2ul of 10X AmpLigase buffer 1ul of 100uM competing oligos each (Solexa_up and Solexa_lo
[gDNA] DF6-9-9: 221ng/ul (5ul needed) foreskin: 153ng/ul (6.5ul needed) CViB: 48.7ng/ul (20.5ul needed) CViF: 77.7ng/ul (13ul needed)
exome probe (with Jan09 1-5,8,9,Mar R2, R3): 7ng/ul (14ul needed)
system setup (all units are in ul):
reagents | DF | foreskin | CviB | CviF |
DNA | 5 | 6.5 | 20.5 | 13 |
probe | 14 | 14 | 14 | 14 |
10x ampligase buffer | 4 | 4 | 4 | 4 |
human Cot-1 DNA | 2.5 | 2.5 | 2.5 | 2.5 |
Solexa_up (100uM) | 1 | 1 | 1 | 1 |
Solexa_lo (100uM) | 1 | 1 | 1 | 1 |
ddH2O | 2.5 | 1 | 7 | 4.5 |
total: | 30 | 30 | 50 | 40 |
95C 5min -> cool down to 60C at 0.01C/sec -> 60C 24 hours -> 55C 24 hours.