Editing
Alice:Exome/2014-1-9
(section)
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=Whole genome library prep (gDNA from Dr. Linzhao Cheng Lab in Johns Hopkins University)= *Received genomic DNA for the 6 samples of BC1 cells targeting the AAVS1 locus by either TALEN or Cas9-gRNA yesterday *All of the samples are ~200ng/ul in 40ul *Sheared at IGM facility in UCSD, program the Covaris for target size of 300 bp List of samples: 1) BC1 p 16+14 (parental) 2) BC1 p16+21 (control) 3) BC1 AAV GFP TALEN c3 p16+21 4) BC1 AAV GFP TALEN c6 p16+21 5) BC1 AAV GFP hCas9 c4 p16+21 6) BC1 AAV GFP hCas9 c16 p16+21 *Kapa Library construction kit for Illumina is used (KK8201) ==End-repair Reactions== Fragmented DNA 100 ul 10X End Repair Reaction Buffer 12 ul End Repair Enzyme Mix 5 ul Keep the tube at ~20°C for 30 minutes. Ampure bead purification (1.6x) and elute in 32.5ul 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 30 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul A Tailing Enzyme 3 ul H2O 12 ul Incubated at 30C for 30min Ampure bead purification (1.8x) and elute in 32.5ul ddH2O ==Adaptor ligation== Prepare 30uM adaptors: 100uM PE-t: 30ul 100uM PE-b: 30ul 10x stoffel buffer: 10ul H2O: 30ul 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 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. ordering high quality HPLC purified adaptor will help reduce self-ligated adaptor sequences, thus eliminate the 125bp chimeric band 5x Kapa Ligation buffer 10ul DNA ligase 5ul DNA adaptor (30uM) 5ul A tailed DNA 30ul Incubate at 20C for 20 minutes purify the product with Ampure beads (1X) and elute in 42ul ddH2O ==PCR amplification== Ligation products 10ul 10uM PCR_F 1ul 10uM PCR_R_index 1ul (designed by Dinh: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Multiplex_primers_for_the_N2-adaptor Link]] 2X Phusion HF MasterMix 25ul H2O 13ul 4 well for each sample 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 60ul total per sample Perform another bead purification (1x) for size selection purpose and elute in 42ul ddH2O total for each sample ==Qubit dsDNA HS Assay== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- align="center" valign="bottom" | width="163" height="12" | # | width="185" | Sample | width="139" | Sample Concentration (ng/ul) |- align="center" valign="bottom" | align="center" height="12" | 1 | bc1_parent_N2-1_seq_lib | align="center" | 16.2 |- align="center" valign="bottom" | align="center" height="12" | 2 | bc1_control_n2-2_seq_lib | align="center" | 21 |- align="center" valign="bottom" | align="center" height="12" | 3 | WGL-talen_C3_N2-3_seq_lib | align="center" | 21 |- align="center" valign="bottom" | align="center" height="12" | 4 | WGL-Talen_C6_N2-4_seq_lib | align="center" | 19.5 |- align="center" valign="bottom" | align="center" height="12" | 5 | WGL-hcas9_C4_N2-5_seq_lib | align="center" | 21.2 |- align="center" valign="bottom" | align="center" height="12" | 6 | WGL-hcas9_C16_N2-6_seq_lib | align="center" | 17.7 |} ==PAGE Gel Quantification== [[File:ZhangLab_2 2014-01-16 12hr 50min.jpg|400px]] ==pooling the libraries for PAGE gel purification== {| {{table}} border=1 style="text-align: center;" | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Sample Concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''amount needed (ul)''' |- | WGL_bc1_parent_N2-1_seq_lib||16.2||10 |- | WGL_bc1_control_n2-2_seq_lib||21||8 |- | WGL-talen_C3_N2-3_seq_lib||21||8 |- | WGL-Talen_C6_N2-4_seq_lib||19.5||8 |- | WGL-hcas9_C4_N2-5_seq_lib||21.2||8 |- | WGL-hcas9_C16_N2-6_seq_lib||17.7||9 |} ==PAGE size selection and purification== *total amount from above is 51 ul, then add 9 ul H2O and 20 ul 6x loading dye to load onto 2 PAGE gel cassettes and 4 lanes total [[File:ZhangLab_2 2014-01-16 16hr 26min-edit.jpg]] size select DNA smear in the range of 300 bp to 700 bp, shear the gel pieces through 0.5 ml tube(pierced with G20 gauge needle) add 450 ul of 1x TE buffer shake in 37C for 1 hour centrifuge the tube and transfer the supernatant with limited amount of gel pieces onto Nanosap columns recover ~420 ul of product and purify with ethanol precipitation ==Quantification and calculation for sequencing== *PAGE gel validation: [[File:ZhangLab_2 2014-01-17 12hr 52min-edit.jpg]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information