Sam:LabNotes/Human/2009-10-25: Difference between revisions
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>Sam Chiang |
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[[Image:sam102609-TBU-4th test.jpg|450px]] [[Image:sam102609-TBU-4th test -post cut.jpg|450px]] | [[Image:sam102609-TBU-4th test.jpg|450px]] [[Image:sam102609-TBU-4th test -post cut.jpg|450px]] | ||
Left: Pre-cutting Right: Post-cutting | |||
NOTE: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting. | NOTE: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting. |
Revision as of 23:31, 29 October 2009
Solexa sequencing library construction - 4th test
Background
- I noticed that I can use human chromosome primer to generate the PCR product for positive control.
Sample preparation
- Prepare two types of postive control with human chro primer P1(270bp) and P11 (130bp)
- Template: Jurket gDNA
- Purify the PCR product using Montage filter (Millipore). Elute in 20uL H2O.
- Results: P1 DNA(270bp): 158.16 ng/uL in 20uL. P11 DNA(130bp): 92.82 ng/uL in 20uL.
Blunting
- Set up 8 reaction with 400ng DNA for starting amount. Add H2O to DNA sample to make the volume needed for reaction.
- Comparing End-it kit(EPI) and Quick blunting kit(NEB)
- Set reactions on cold block
End-it Kit - test on sample B1, B2, B3, B4 1rxn 4.5rxn DNA 28.0 - 10X blunting buf. 4.0 18.0 1mM dNTP mix 4.0 18.0 Blunting Enzyme 4.0 18.0 ------------------------------------ 40.0 54.0 54/4.5=12 --- DNA 28uL
Quick blunting Kit - test on B5, B6, B7, B8 1rxn 4.5rxn DNA 18.0 - 10X blunting buf. 2.5 11.25 1mM dNTP mix 2.5 11.25 Blunting Enzyme 2.0 9.0 ------------------------------------ 25.0 31.5 31.5/4.5=7 --- DNA 18L
- Mix the reaction by vortexing 5sec. Incubate at RT for 30min.
- No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB.
A-tailing
- Comparing two Klenow(-) enzymes
- Exo-Minus Klenow DNA polymerase(EPICENTRE) - for A1, A3, A5, A7
- Klenow fragment (3'->5' exo-)(NEB) - for A2, A4, A6, A8
- Set reactions on cold block
1rxn 4.5rxns(EPI) Blunted DNA 30 - 10X buf. 4 18 (10X Knenow buf.) 1mM dATP 6 27 Enzyme 2 9 ------------------------------------------ 40 54/4.5=12 - DNA 30uL
1rxn 4.5rxn(NEB) Blunted DNA 28 10X buf. 4 18(10X buf."2") 1mM dATP 6 27 Enzyme 2 9 ------------------------------------------ 40 54/4.5=12 - DNA 28uL
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
- No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.
Ligation
- T4 ligation kit (Enzymatic)
- Only use the Y-adaprot I made (1st made Y-adaptor)
- Adaptor:Insert calculation
- Assume that there is only 50% DNA left after all of prevous steps (Note: There is no size-selection in this test).
- DNA before ligation: 200ng of 270bp and 200ng of 130bp (Starting amount: 400ng)
- 200/(660x270)x10^6= 1122.33nM = 1.122uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 11.23uM ~ 56.11uM Y-adaptor needed.
- By using 50:1 for Adaptor:Insert, the Adaptor should be 63131nM = 63uM -> 3uL of 20uM Y-adaptor
- 200/(660x130)x10^6= 2331.00nM = 2.331uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 23.31uM ~ 116.55uM Y-adaptor needed.
- By using 50:1 for Adaptor:Insert, the Adaptor should be 63131nM = 63uM -> 3uL of 20uM Y-adaptor
9rxn A-tailed DNA 10 - 2X rapid liagas buf. 15 135.0 20uM Y-adaptor 3 27.0 T4 ligase 2 18.0 --------------------------------- 30 180.0/9=20uL --- DNA 10 uL
- Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
- No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 10uL EB (All of 10uL ligation will used for TBU-PAGE size selection).
PAGE TBU gel size selection
- A 5-well TBU gel is recommanded since it has large capacity for sample loading.
- Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye
- Ladder: 0.5uL 25bp ladder + 9uL H2O + 10uL 2X TBU-loading dye
- Heat denaturing: 75C 15min -> 1min on cool block
- Flush TBU gell well, pre-run 10min at 200V
- Flush TBU gell well. Load denatured samples and ladders(20uL). Run at 200V for 30min.
- Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam102609-TBU-4th test.jpg File:Sam102609-TBU-4th test -post cut.jpg Left: Pre-cutting Right: Post-cutting NOTE: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting.