Alice:LabNotes/2011-4-4: Difference between revisions

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>Zsakura2
 
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  purify the products with Qiaquick columns and elute in 40ul ddH2O
  purify the products with Qiaquick columns and elute in 40ul ddH2O
*Nanodrop result:
*Nanodrop result:
*HGPS: 69.2ng/ul * 40ul
*HGPS-2: 69.2ng/ul * 40ul
*[[File:ZhangLab_2 2011-04-05 18hr 05min.jpg]]
*[[File:ZhangLab_2 2011-04-05 18hr 05min.jpg]]



Latest revision as of 21:19, 10 May 2011

Prepare genomic DNA for hybridization (for use with Nimblegen's kit)[edit]

  • received a new batch of cell pellet (HGPS only) on 3/31/2011, finished the DNA extraction on 4/1/2011
  • sheared the 3ug of DNA at UCSD Moores Cancer Center (Frazer Lab)
  • sample to be processed: HGPS

End-repair Reactions[edit]

Fragmented DNA                             85 ul
10X End Repair Reaction Buffer             11 ul
End Repair Enzyme Mix                      4  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.
Nanodrop result (40ul total for each):
HGPS: 46.6ng/ul

A-Tailing reactions[edit]

Blunt-end DNA                             40 ul
10X dA-Tailing Reaction Buffer (10X)       5 ul
Klenow Fragment (3’-5’ exo-)               3 ul
H2O                                        2 ul
Incubated at 37C for 30min
purified the products with Qiaquick column and elute in 30ul ddH2O
Nanodrop result (30ul total for each):
HGPS: 69.4ng/ul

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. target:adaptor molar ratio is 1:15
A-tailed DNA:        30ul
5x ligation buffer:  12ul
20uM Y adaptor:      11ul
Quick T4 Ligase:      5ul
Incubate at room temperature for 30 minutes
purify the product with Qiaquick columnes and elute in 40ul ddH2O

PCR amplification[edit]

10ul of ligated DNA for each well, and a total of 4 well
Ligation products                 10ul  
100uM PCR_F                       0.2ul        
100uM PCR_R                       0.2ul
2X phusion HF master mix          50ul     
H2O                               40ul       
PCR program: 98 °C 30sec  -> 7 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. 
purify the products with Qiaquick columns and elute in 40ul ddH2O

Results[edit]

  • The yield of the last PCR step is very low compare to the previous whole-genome libraries that I have prepared. However, the result agrees with the whole-genome library of the same sample I prepared on 3/28/2011. Although the results aren't as good as the previous libraries that I have prepared, but we should still be able to get useful information after sequencing. Therefore I will combine the library from 3/28/2011 with this batch to do liquid capture.

Hybrid Capture using Nimblegen kit (4/6/2011)[edit]

  • 3/28/2011 batch: HGPS- 34ng/ul * 60ul
  • 4/4/2011 batch: HGPS- 69.2ng/ul * 40ul
  1. Add 5μl of 1mg/ml COT DNA and 2.5μg of amplified sample library to a new 1.5ml tube.
  2. Add 10μl of each 100μM PE-HE1 and PE-HE2 Oligos to the amplified sample library plus COT DNA. (PE-HE1=PCR_F, PE-HE2=PCR_R)
  3. Close the tube’s lid and make a hole in the top of the tube’s cap with an 18 - 20 gauge or smaller needle. (Note: The hole in the top of the tube’s cap is a precaution to suppress contamination in the SpeedVac.)
  4. Dry the amplified sample library/COT DNA/PE-HE Oligos in a SpeedVac on high heat (60°C). (Note: This step may take 30 minutes or longer. To minimize drying time, dry COT DNA ahead of time and then add the amplified sample library and PE- HE Oligos. Denaturation of the DNA with high heat is not problematic after linker ligation because the hybridization utilizes single-stranded DNA.)
  5. To each dried-down amplified sample library/COT DNA/PE-HE Oligos, add 7.5μl of 2X SC Hybridization Buffer and 3μl of SC Hybridization Component A
  6. Cover the hole in the tube’s cap with a sticker or small piece of laboratory tape. Vortex the amplified sample library/COT DNA/PE-HE Oligos plus Hybridization Cocktail (2X SC Hybridization Buffer + SC Hybridization Component A) for 10 seconds and centrifuge at maximum speed for 10 seconds.
  7. Place the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail in a 95°C heat block for 10 minutes to denature the DNA.
  8. Centrifuge the amplified sample library/COT DNA/PE-HE/Hybridization Cocktail at maximum speed for 10 seconds at room temperature.
  9. Transfer the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail to the 4.5μl aliquot of Exome Library in a 0.2ml PCR tube prepared in Chapter 2 (entire volume can also be transferred to a 96-well PCR plate). Vortex for 3 seconds and centrifuge at maximum speed for 10 seconds.
  10. Incubate in a thermocycler (with heated lid turned on at 57C) at 47°C for 72 hours.