Alice:LabNotes/2010-3-8: Difference between revisions
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| human Cot-1 DNA||2.5||2.5||2.5 | | human Cot-1 DNA||2.5||2.5||2.5 | ||
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| Solexa_lo (100uM)||1||1||1 | | Solexa_lo (100uM)||1||1||1 | ||
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| ddH2O|| | | ddH2O||6||6||4.5 | ||
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Revision as of 17:08, 9 March 2010
Prepare genomic DNA for hybridization
- samples to be prepared: CVF-gDNA, CViB-gDNA,
- two sheared gDNA received from Harvard: CD1 PGP1 ips P16 and PGP1F 8 gDNA
End-repair Reactions
Fragmented DNA 85 ul 10X End Repair Reaction Buffer 10 ul End Repair Enzyme Mix 5 ul Keep the tube at room temperature (~20°C) for 30 minutes. Purify with Qiaquick column and elute in 40ul ddH2O Note: for blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
A-Tailing reactions
Blunt-end DNA 37 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul Klenow Fragment (3’-5’ exo-) 3 ul H2O 5 ul Incubated at 37C for 30min purified the products with Qiaquick column and elute in 40ul ddH2O
adaptor ligation
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 5X Quick ligase buffer 10 ul Quick Ligase 3 ul H2O 24 ul
Incubate at room temperature for 15 minutes purify the product with Agencourt AMpure kit and elute in 40ul ddH2O
PCR
Ligation products 5ul (8 well for each set, total of 80 well) 100uM Solexa_PCR_up 0.2ul 100uM solexa_PCR-lo 0.2ul 2X phusion HF master mix 50ul 50X SYBR Green I 0.4ul H2O 45ul PCR program: 98 °C 30sec -> 13 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. purify the products with Qiagen Qiaquick columns and elute in 40ul ddH2O
Biotin-labeled probe capture
- gDNA samples of DF and foreskin will be used, and the preparation protocol can be found under labnote 2-1-2010
working buffer preparation
1x binding buffer (following Nimblegen setup): 1M NaCl: 1000ul 10mM Tris-HCl (pH ~7): 25ul 1mM EDTA: 5ul ddH2O: 1470ul total: 2.5ml
Hybrid selection
Mix the following to 20ul total volume: 1ug ligated DNA with 200ng 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)
exome probe set 1: w/o PAGE selection (with Jan09 1-5,8,9,Mar R2, R3): 7ng/ul (28ul needed) exome probe set 2: w/ PAGE selection (Jan09 1-5,8,9): 7ng/ul (28ul)
system setup (all units are in ul):
reagents | DF | DF(for exome probe set2) | foreskin |
DNA | 5 | 5 | 6.5 |
probe | 28 | 28 | 28 |
10x ampligase buffer | 5 | 5 | 5 |
human Cot-1 DNA | 2.5 | 2.5 | 2.5 |
Solexa_up (100uM) | 1 | 1 | 1 |
Solexa_lo (100uM) | 1 | 1 | 1 |
ddH2O | 6 | 6 | 4.5 |
total: | 50 | 50 | 50 |
95C 5min -> cool down to 60C at 0.01C/sec -> 60C 24 hours -> 50C 24 hours.
Prepare the Streptavidin Dynabeads
a. Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear b. add twice the volume of the beads of 1x binding buffer, place the tube on magnet and remove the liquid when the solution becomes clear c. wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA) d. resuspend the beads in 200ul 1x binding buffer, warm up to 45C. c. Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min, take 1/2 and go on with no wash. d. Remove the liquid from the beads with a magnet. e. Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 45C. f. After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min. g. Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5. h. Purifythe 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB.
Post-capture PCR
a.Set up a 400ul reaction with Phusion High-Fidelity PCR master mix: 2x Phusion master mix: 200ul 100uM PCR_F 2ul 100uM PCR_R 2ul 50X SYBG I 3.2ul Captured DNA 30ul H2O 163ul b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. Terminate the reaction when the amplification curves approach to the plateau.
Measurement of Enrichment using qPCR on biotin-labeled probe capture sample
- The qPCR assay is used to estimate relative fold-enrichment by measuring the relative abundance of control targets in amplified sample library and amplified captured DNA to determine whether the capture was successful.
Preparation
NSC qPCR assay name | primer sequence 5'->3' | Tm ( C) | length |
NSC-0237 | F: CGCATTCCTCATCCCAGTATG | 81.15 | 80bp |
R: AAAGGACTTGGTGCAGAGTTCAG | |||
NSC-0247 | F: CCCACCGCCTTCGACAT | 81.03 | 74bp |
R: CCTGCTTACTGTGGGCTCTTG | |||
NSC-0268 | F: CTCGCTTAACCAGACTCATCTACTGT | 78.99 | 75bp |
R: ACTTGGCTCAGCTGTATGAAGGT | |||
NSC-0272 | F: CAGCCCCAGCTCAGGTACAG | 82.23 | 71bp |
R: ATGATGCGAGTGCTGATGATG | |||
- Dilute the NSC assay forward and reverse primers to 2μM.
- Dilute sufficient amounts of amplified sample library and amplified captured DNA to a concentration of 5ng/μl in PCR grade water for use as qPCR templates
PCR amplification
PCR grade water 19.7ul NSC Assay forward primer (2uM) 1ul NSC Assay reverce priimer (2uM) 1ul SYBR Green (50X) 1ul template (5ng/ul) 3.3ul KAPA 2x master mix 25ul 95C 3min -> (95C 3sec -> 60C 20sec -> 72C 3sec) x40 -> 72C 3min -> 15C hold