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.