Sam:LabNotes/Human/2009-10-25: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang (New page: ='''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 prep...) |
>Sam Chiang No edit summary |
||
Line 36: | Line 36: | ||
==A-tailing== | ==A-tailing== | ||
*Exo-Minus Klenow DNA polymerase(EPICENTRE) | *Comparing two Klenow(-) enzymes | ||
*Klenow fragment (3'->5' exo-)(NEB) | **Exo-Minus Klenow DNA polymerase(EPICENTRE) - for A1, A3, A5, A7 | ||
**Klenow fragment (3'->5' exo-)(NEB) - for A2, A4, A6, A8 | |||
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) 4.5rxn(EPI) | |||
1rxn | |||
Blunted DNA 20 | Blunted DNA 20 | ||
H2O 8 20 36 | H2O 8 20 36 |
Revision as of 18:00, 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)
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
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) 4.5rxn(EPI) Blunted DNA 20 H2O 8 20 36 10X buf. 4 10(10X buf."2") 18 (10X Knenow buf.) 1mM dATP 6 15 27 Enzyme 2 5 9 ------------------------------------------ 40 50/2.5=20 90/4.5=20 --- DNA 20uL
- Transfer 20uL blunted DNA into PCR tubes
- Transfer 20uL master mix into reactions
- Mix the reaction by vortexing 5sec. Incubate at 37 for 30min -> 75C 20min (Inactivation)
- Purify the A-tailed DNA with QIAquick column. Elute in 20uL H2O.
Ligation
- T4 ligation kit (Enzymatic)
- Only try the positive control in two different sources of Y-adaptor (Sam-made or Jie-made)
- Adaptor:Insert calculation
- The original positive control DNA is 358 ng/uL by Nanodrop(H2O blank)
- Assume we use 358ng (1uL) in the begining. After two QIAquick washing => 250ng/rxn
- The sample was seperated into two => 125ng/rxn. If the sample is around 150bp then 125/(660x150)x10^6=2525nM --- Insert
- By using 50:1 for Adaptor:Insert, the Adaptor should be 63131nM = 63uM -> 3uL of 20uM Y-adaptor
- Exp. design
- A5-1, A5-2, A6-1, A6-2 (A5-1 and A6-1 using Y-adaprot from Sam; A5-2 and A6-2 using Y-adaprot from Jie)
2.5rxn DNA 10 - 2X rapid liagas buf. 15 37.5 T4 ligase 2 5.0 20uM Y-adaptor 3 7.5 -------------------------------- 30 50.0/2.5=20uL --- DNA 10 uL Incubate at RT for 15min. Immediately followed by QIAquick purification. Elute in 20uL H2O
PAGE TBU gel evaluation
- Sample denaturing: Mixed in PCR tube -> 75C 10min -> immediately put on ice for 1min
- Ladder: 6uL Low mass(prediluted) + 3uL 6X loading dye.
- Sample: 3uL sample + 3uL 0.5X TBE + 3uL 6X loading buf.
Results
File:Sam102309-TBU.jpg File:Sam102309-TBE.jpg From left to right: 5-1, 5-2, 6-1, 6-2
- The TBU is not successful since the sample flow out during the loading. The low mass ladder is strange for the position of 100bp band. There shouldn't be a huge seperation between 100bp and 200bp bands.
- I ran the same samples on TBE gel. A very dim band could be found above 400bp band (difficult to be seen on the picture). If Alice's assumption is right that Y-adaptor ligated DNA will be hampered during gel electrophoresis migration, then these dim bands might be our target (expected size: 150bp + 100bp (Y-adaptor) = 250bp
File:Sam102509-TBU-correct.jpg From left to right: 5-1, 5-2, 6-1, 6-2
- A new TBU-PAGE was run with corrected procedures
- 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
- Load denatured samples and ladders(20uL). Run at 200V for 30min.
- Stained with 3uL SYBR-Gold in TBE for 5min
- With a longer exposure time, a smear band could be found around 225~250bp. It is possible the ligated insert. Unfortunately, the band is weak to be cut for qPCR testing.
- I noticed that the weak band could be cuased by the low concentration of ligation product.
- Original amount: 358ng/rxn
- Estimated amount before ligation(50%): 179ng/rxn
- Divided into two for two ligations: 89.5ng/rxn
- Ligation result 89.5ng/30uL => 2.98 ng/uL
- TBU loading 10uL => 30ng/well