Sam:LabNotes/Human-Chip-bisulfite-Seq/2009-11-19: Difference between revisions
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**Wash the pellet with 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant. | **Wash the pellet with 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant. | ||
**Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O. | **Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O. | ||
='''qPCR amplification of Solexa library (Human chro MDA samples)'''= | |||
==qPCR amplification== | |||
*Only use half of amount (10uL)of ligation product for qPCR amplification. | |||
*Solexa lib. templates: | |||
**No.10, N10, No.12, N12 (N10 and N12 are samples made on 10/30/'09, with addition of DNA pol.I) | |||
*Primers - from Dr. Zhang | |||
**(Yellow cap, Tm=70.7, MW=17617.4) Solexa_PCR_upH (10uM) | |||
**(Yellow cap, Tm=72.4, MW=18710.1) Solexa_PCR_PE_lo_H (10uM) | |||
Master mix | |||
4.5 rxn | |||
H2O 31.6 142.2 | |||
Template 10.0 - | |||
Solexa_PCR_upH (10uM) 4.0 18.0 | |||
Solexa_PCR_PE_lo_H (10uM) 4.0 18.0 | |||
SYBR 50X 0.4 1.8 | |||
2X Fusion enzyme mix(Enzymatics) 50.0 225.0 | |||
----------------------------------------------- | |||
100.0 405.0/4.5=90 --- 10uL template | |||
*Using two annealing temp program for amplification | |||
**Use 5 cycles for the first annealing temp(64C). | |||
**Use 16 cycles for the second annealing temp(72C). | |||
Line1: 98C 30sec | |||
Line2: 98C 10sec | |||
Line3: 64C 20sec | |||
Line4: 72C 20sec | |||
Line5: Read plate | |||
Line6: Goto line 5 for 4 more times | |||
Line7: 98C 10sec | |||
Line8: 72C 20sec | |||
Line9: Read plate | |||
Line10: Goto line 7 for 15 more times | |||
Line11: 72C 5min | |||
Line12: 15C forever | |||
END | |||
*Tterminate/pause the reaction at annealing step (72C) when it's closing to saturation stage. | |||
[[Image:sam110209-qPCR 1-5cycle.jpg|600px]] [[Image:sam110209-qPCR 6-8 cycle.jpg|600px]] | |||
The N10 reaction stopped at 5th cycle. The No.10, No.12 and N12 stopped at 8th cycle due to the slower increasement of signal. | |||
*Check the qPCR product (3uL) on TBE gel to evaluate the library results. | |||
[[Image:sam110209 - TBE validation of amplified lib.jpg|400px]] | |||
*The expected size of libraries are: | |||
**200~300bp + Y-adaptor (100bp)= 300~400bp | |||
*Except N10, all of the reactions seem over-amplified with the larger band size ~800bp | |||
*I should use the left samples (the other 10uL) to repeat this amplification again. | |||
==Repeat qPCR amplification== | |||
*Use the left ligation product (10uL) for qPCR amplification. | |||
*Solexa lib. templates: | |||
**No.12 and N12 | |||
*Primers - from Dr. Zhang | |||
**(Yellow cap, Tm=70.7, MW=17617.4) Solexa_PCR_upH (10uM) | |||
**(Yellow cap, Tm=72.4, MW=18710.1) Solexa_PCR_PE_lo_H (10uM) | |||
1rxn | |||
H2O 31.6 | |||
Template 10.0 | |||
Solexa_PCR_upH (10uM) 4.0 | |||
Solexa_PCR_PE_lo_H (10uM) 4.0 | |||
SYBR 50X 0.4 | |||
2X Fusion enzyme mix(Enzymatics) 50.0 | |||
--------------------------------------- | |||
100.0 | |||
*Using two annealing temp program for amplification | |||
*Terminate/pause the reaction at annealing step (72C) when it's closing to saturation stage. | |||
*Immediately transfer 50uL of reaction into a fresh PCR tube for an extra cycle amplification in Chromo4. | |||
[[Image:sam110209-qPCR new no12 and N12.jpg|500px]] | |||
[[Image:sam110209-TBE-gel check new 12.jpg|350px]] | |||
*The N12 sample and 6-cycle amplification showed the strong band at the expected size (~400bp). The rest of lib reaction showed no obivous band probably due to the low concentration. | |||
==Product purification== | |||
*Purify the amplified library N10 and N12 with QIAquick column | |||
**Take 20uL sample and eluted with 15uL EB. | |||
*Gave the purified library to Alan for Solexa sequencing. |
Revision as of 08:02, 22 November 2009
Low-input DNA Solexa seq. library construction test using dUTP-carrier
Background
- It has been proved that dUTP-carrier can be digested by USEr enzyme and S1 endonuclease.
- I am testing if dUTP-carrier can protect low-input DNA in library construction.
- The low-input DNA sample were obtained from the NlaIII digested Jurket DNA (25ng/uL) from 10/23/'09 exp.
Carrier preparation
- EtOH/NaOAC purify the dUTP-PCR product made on 11/17/'09
- 100uL dUTP-PCR product + 250uL 100%EtOH + 10uL 3M NaOAc + 1.5uL Glycoblue -> -80C precipitation -> 750uL 75% EtOH washing
- E1U-e: 386.52 ng/uL 1ug=2.58uL
- E3U-e: 181.06 ng/uL 1ug=5.52uL
Sample preparation
- NlaIII digested Jurket DNA 500ng/20uL=25ng/uL
50ng-Carrier 100ng-Carrier 200ng-Carrier DNA sample 2.00uL 4.00uL 8.00uL E1U-e(1ug) 2.58 2.58 2.58 H2O 23.42 21.42 17.42 -------------------------------------------------------- 28.00 28.0 28.00
50ng-Carrier 100ng-Carrier 200ng-Carrier DNA 2.00uL 4.00uL 8.00uL E3U-e(1ug) 5.52 5.52 5.52 H2O 20.48 18.48 14.48 -------------------------------------------------------- 28.00 28.00 28.0
Blunting
- End-it Kit (EPICENTRE)
- Set reactions on cold block
1rxn 7rxn DNA 28.0 - 10X blunting buf. 4.0 28.0 dNTP mix 4.0 28.0 Blunting Enzyme 4.0 28.0 ------------------------------------ 40.0 84.0 84/7=12 --- DNA 28uL Add the DNA pol.I (E.coli) 1uL on the top of reaction
- Mix the reaction by vortexing 5sec. Incubate at RT for 30min.
- During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (A-tailing)
- No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB.
NOTE: The reaction B3-2 and B3-3 maybe mis-labeled in this step.
A-tailing
- Exo-Minus Klenow DNA polymerase(EPICENTRE)
- Set reactions on cold block
- Prepare the fresh dilution of 100mM dATP to 1mM dATP
1rxn 7rxns DNA 30 - 10X Knenow buf. 4 28 1mM dATP 6 42 Klenow enzyme 2 14 ------------------------------------------ 40 84/7=12 - DNA 30uL
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
- During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (Ligation)
- No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.
Ligation
- T4 ligation kit (Enzymatic)
- Set reactions on cold block
- Adaptor:Insert calculation
- Assume that 50% DNA left after all of previous steps (no sample size-selpection). (1ug + X ng)/2=500ng (X=sample amount -> ignored in the calculation)
- DNA before ligation: 500ng of 250bp
- 500/(660x250)x10^6/1000 = 3.03uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 30.3uM ~ 151.5uM Y-adaptor needed.
- I decided to use 3uL of 20uM Y-adaptor which is in between 10:1 and 50:1 rule.
7rxn A-tailed DNA 11 - 2X rapid liagas buf. 15 105.0 20uM Y-adaptor 3 21.0 T4 ligase 2 14.0 ---------------------------------- 30 140.0/7 = 20uL --- DNA 10uL
NOTE: In order to increase production, I decided to add all of the DNA (12uL) eluted from previous step.
- Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
- No inactivation needed, but purify the ligated DNA with QIAquick column. Elute in 10uL EB.
TBU-PAGE size selection
- TBU-PAGE condition: 5-well TBU. Run at 200V, 30min
- Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye
- Ladder: 0.5uL 25bp ladder + 9uL 0.5X TBE + 10uL 2X TBU-loading dye
- Heat denaturing: 75C 15min -> on cool block (>1min)
- Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam111909-tbu gel 1.jpg File:Sam111909-tbu gel 1-post cut.jpg Left: Pre-cutting Right: Post-cutting File:Sam111909-tbu gel 3.jpg File:Sam111909-tbu gel 3-post cut.jpg Left: Pre-cutting Right: Post-cutting
Recovery of size selected DNA
- Gel-cut slice was sheared by centrifuging at 14000 rpm at RT, 3min using a 22G needle punched 0.5mL tube.
- Sheared gel was soaked in 400uL clean 1X TE and incubate at 37C shaker (>2hr or overnight). shaking at the lowest speed.
- Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant onto a Nanosep column filter.
- Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through.
- Perform DNA purification with EtOH/NaOAc method.
- Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue. Incubate at -80C for 25min (or longer)
- Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 25min. Remove the supernatant.
- Wash the pellet with 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant.
- Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
qPCR amplification of Solexa library (Human chro MDA samples)
qPCR amplification
- Only use half of amount (10uL)of ligation product for qPCR amplification.
- Solexa lib. templates:
- No.10, N10, No.12, N12 (N10 and N12 are samples made on 10/30/'09, with addition of DNA pol.I)
- Primers - from Dr. Zhang
- (Yellow cap, Tm=70.7, MW=17617.4) Solexa_PCR_upH (10uM)
- (Yellow cap, Tm=72.4, MW=18710.1) Solexa_PCR_PE_lo_H (10uM)
Master mix 4.5 rxn H2O 31.6 142.2 Template 10.0 - Solexa_PCR_upH (10uM) 4.0 18.0 Solexa_PCR_PE_lo_H (10uM) 4.0 18.0 SYBR 50X 0.4 1.8 2X Fusion enzyme mix(Enzymatics) 50.0 225.0 ----------------------------------------------- 100.0 405.0/4.5=90 --- 10uL template
- Using two annealing temp program for amplification
- Use 5 cycles for the first annealing temp(64C).
- Use 16 cycles for the second annealing temp(72C).
Line1: 98C 30sec Line2: 98C 10sec Line3: 64C 20sec Line4: 72C 20sec Line5: Read plate Line6: Goto line 5 for 4 more times Line7: 98C 10sec Line8: 72C 20sec Line9: Read plate Line10: Goto line 7 for 15 more times Line11: 72C 5min Line12: 15C forever END
- Tterminate/pause the reaction at annealing step (72C) when it's closing to saturation stage.
File:Sam110209-qPCR 1-5cycle.jpg File:Sam110209-qPCR 6-8 cycle.jpg The N10 reaction stopped at 5th cycle. The No.10, No.12 and N12 stopped at 8th cycle due to the slower increasement of signal.
- Check the qPCR product (3uL) on TBE gel to evaluate the library results.
File:Sam110209 - TBE validation of amplified lib.jpg
- The expected size of libraries are:
- 200~300bp + Y-adaptor (100bp)= 300~400bp
- Except N10, all of the reactions seem over-amplified with the larger band size ~800bp
- I should use the left samples (the other 10uL) to repeat this amplification again.
Repeat qPCR amplification
- Use the left ligation product (10uL) for qPCR amplification.
- Solexa lib. templates:
- No.12 and N12
- Primers - from Dr. Zhang
- (Yellow cap, Tm=70.7, MW=17617.4) Solexa_PCR_upH (10uM)
- (Yellow cap, Tm=72.4, MW=18710.1) Solexa_PCR_PE_lo_H (10uM)
1rxn H2O 31.6 Template 10.0 Solexa_PCR_upH (10uM) 4.0 Solexa_PCR_PE_lo_H (10uM) 4.0 SYBR 50X 0.4 2X Fusion enzyme mix(Enzymatics) 50.0 --------------------------------------- 100.0
- Using two annealing temp program for amplification
- Terminate/pause the reaction at annealing step (72C) when it's closing to saturation stage.
- Immediately transfer 50uL of reaction into a fresh PCR tube for an extra cycle amplification in Chromo4.
File:Sam110209-qPCR new no12 and N12.jpg
File:Sam110209-TBE-gel check new 12.jpg
- The N12 sample and 6-cycle amplification showed the strong band at the expected size (~400bp). The rest of lib reaction showed no obivous band probably due to the low concentration.
Product purification
- Purify the amplified library N10 and N12 with QIAquick column
- Take 20uL sample and eluted with 15uL EB.
- Gave the purified library to Alan for Solexa sequencing.