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.