Sam:LabNotes/Human/2009-10-25: Difference between revisions

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Line 20: Line 20:
  DNA                  28.0      -
  DNA                  28.0      -
  10X blunting buf.      4.0    18.0
  10X blunting buf.      4.0    18.0
  1mM dNTP mix           4.0    18.0
  dNTP mix               4.0    18.0
  Blunting Enzyme        4.0    18.0
  Blunting Enzyme        4.0    18.0
  ------------------------------------
  ------------------------------------
Line 29: Line 29:
  DNA                  18.0      -
  DNA                  18.0      -
  10X blunting buf.      2.5    11.25
  10X blunting buf.      2.5    11.25
  1mM dNTP mix           2.5    11.25
  dNTP mix               2.5    11.25
  Blunting Enzyme        2.0      9.0
  Blunting Enzyme        2.0      9.0
  ------------------------------------
  ------------------------------------
Line 50: Line 50:
  ------------------------------------------
  ------------------------------------------
               40      54/4.5=12 - DNA 30uL
               40      54/4.5=12 - DNA 30uL


               1rxn    4.5rxn(NEB)  
               1rxn    4.5rxn(NEB)  
Line 62: Line 61:
*Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
*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.
*No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.


==Ligation==
==Ligation==
Line 68: Line 66:
*Only use the Y-adaprot I made (1st made Y-adaptor)
*Only use the Y-adaprot I made (1st made Y-adaptor)
*Adaptor:Insert calculation
*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).
**Assume that there is only 50% DNA left after all of previous steps (Note: There is no size-selection in this test).
**DNA before ligation: 200ng of 270bp and 200ng of 130bp (Starting amount: 400ng)
**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.
**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.
Line 84: Line 82:
   
   
*Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
*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).
*No inactivation needed, but purify the ligated DNA with QIAquick column. Elute in 10uL EB (All of 10uL ligation will used for TBU-PAGE size selection).


==PAGE TBU gel size selection==
==TBU-PAGE size selection==
*A 5-well TBU gel is recommanded since it has large capacity for sample loading.
*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
**Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye
**Ladder: 0.5uL 25bp ladder + 9uL H2O + 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
**Heat denaturing: 75C 15min -> 1min on cool block
**Flush TBU gell well, pre-run 10min at 200V
**Flush TBU gel well. Pre-run 10min at 200V
**Flush TBU gell well. Load denatured samples and ladders(20uL). Run at 200V for 30min.
**Flush TBU gel well again. Load denatured samples and ladders(20uL). Run at 200V for 30min.
**Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
**Stained with 3uL SYBR-Gold in 0.5X TBE for 5min


Line 110: Line 108:
*Perform DNA purification with EtOH/NaOAc method.
*Perform DNA purification with EtOH/NaOAc method.
**Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue.
**Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue.
**Gentaly inveted 20times. Incubate at -80C for 25min (or longer)
**Gentaly invert the tube 20times. Incubate at -80C for 25min (or longer)
**Centrifuge at 4C, 10,000xg for 25min. Remove the supernatant(pour + 20p tips). Wash the pellet with 75% EtOH.
**Centrifuge at 4C, 10,000xg for 25min. Remove the supernatant(pour + 20p tips). Wash the pellet with 75% EtOH. Gentaly invert the tube 20times.
**Centrifuge at 4C, 10,000xg for 5min. Remove the supernatant(pour + 20p tips). Air dry the pellet in PCR hood (~10min).
**Centrifuge at 4C, 10,000xg for 5min. Remove the supernatant(pour + 20p tips). Air dry the pellet in PCR hood (~10min).
**Elute the DNA in 20uL H2O.
**Elute the DNA in 20uL H2O.


==qPCR amplification==
==qPCR amplification==
*Only use helf of amount (10uL)for qPCR amplification.
*Only use half of amount (10uL)for qPCR amplification.
*Comparing primers (primer from Alice vs. primers I ordered)
*Comparing primers (primer from Alice vs. primers I ordered)
  Master mix - Sam's primer mix - for L1, L3, L5, L7, N5
  Master mix - Sam's primer mix - for L1, L3, L5, L7, N5
Line 139: Line 137:
                                 100.0  495.0/5.5=90 --- 10uL template
                                 100.0  495.0/5.5=90 --- 10uL template


*Using the new program modifed based on Dr.Zhang's program
*Using the new program modifed based on Dr.Zhang's program (posted on 9-21-'09)
**Use 5 cycles for the first annealing temp(64).
**Use 5 cycles for the first annealing temp(64).
**Use 16 cycles for the second annealing temp(72C).
**Use 16 cycles for the second annealing temp(72C).
Line 159: Line 157:
*Usually the reaction will be close to saturation status around 7~8 cycles. Press the skip buttom to jump to final annealing step when it's closing to saturation stage.
*Usually the reaction will be close to saturation status around 7~8 cycles. Press the skip buttom to jump to final annealing step when it's closing to saturation stage.
   
   
*Check the qPCR product (2~3uL) on TBE gel to evaluate the library results.
*Check the qPCR product (3uL) on TBE gel to evaluate the library results.
  [[Image:sam102709-TBE Solexa lib qPCR results-2.jpg|400px]]
  [[Image:sam102709-TBE Solexa lib qPCR results-2.jpg|400px]]
*The library sizes are as expected. The missed L6 sample was shown on the right size on N6 sample (The additional cutting for L6 sample)
**270bp + Y-adaptor = 370bp; 130bp + Y-adaptor = 230bp
*The primer mix I made showed a stronger primer remains (~25bp) on the gel.
*Little smear were shown on L4 and L8 samples which may be caused by a over amplification.
==Take home messages==
*Elute DNA in EB after QIAquick washing is ok. (Not need to be eluted in H2O)
*Y-adaptor I made is working.
*All of the enzymatic reactions should be set up on cold block
*No inactivation step is needed in each reaction. Direct using QIAquick washing to terminate the enzymatic reaction.
*For the short insert (<200bp), the migration of ligation product on PAGE gel might be biased by the Y-adaptor's sturcture. Therefore a denatured PAGE (TBU) gel is recommanded for the size selection of ligation products. Using 5-well comb TBU is recommanded in order to prevent sample over-loading problem.
*Sample concentration is a key factor too. Use whole amount of product for any following step. The starting amount could be as low as 400ng if there is no size selection after sample shearing.

Latest revision as of 08:49, 9 November 2009

Solexa sequencing library construction - 4th test[edit]

Background[edit]

  • I noticed that I can use human chromosome primer to generate the PCR product for positive control.

Sample preparation[edit]

  • 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[edit]

  • 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
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
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[edit]

  • 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[edit]

  • 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 previous 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 ligated DNA with QIAquick column. Elute in 10uL EB (All of 10uL ligation will used for TBU-PAGE size selection).

TBU-PAGE size selection[edit]

  • 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 gel well. Pre-run 10min at 200V
    • Flush TBU gel well again. 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

NOTE1: I miscalculate the band size of L5 and L6 samples in the beginning, but I added those part by additional cutting.
NOTE2: The gel seems overloading. It is suggested that no more than 100ng/well should be loaded in 12-well PAGE.

Size selected DNA recovery from TBU-PAGE[edit]

  • Prepare gel shearing assemblies by placing a 0.5mL tube (punched a hole in bottom using G22 needle) in a 1.5-mL tube.
  • Transfer the sliced gel in 0.5mL tubes. Centrifuge at 14000 rpm at RT for 3min.
  • After centrifuge, remove the 0.5-mL tubes. Add 500uL 1X TE with the sheared gel in 1.5-mL tubes. Transfer the mixture to a fresh 1.5-mL tube.
  • Incubate the gel-TE mixture at 37C for 1hr on the shaker (shaking at lowest speed)
  • Centrifuge the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. The gel debris will be pellet. Transfer the supernatant into a Nanosep column filter.
  • Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. The tiny gel debris will remain on the filter. Collect the flow through.
  • Perform DNA purification with EtOH/NaOAc method.
    • Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue.
    • Gentaly invert the tube 20times. Incubate at -80C for 25min (or longer)
    • Centrifuge at 4C, 10,000xg for 25min. Remove the supernatant(pour + 20p tips). Wash the pellet with 75% EtOH. Gentaly invert the tube 20times.
    • Centrifuge at 4C, 10,000xg for 5min. Remove the supernatant(pour + 20p tips). Air dry the pellet in PCR hood (~10min).
    • Elute the DNA in 20uL H2O.

qPCR amplification[edit]

  • Only use half of amount (10uL)for qPCR amplification.
  • Comparing primers (primer from Alice vs. primers I ordered)
Master mix - Sam's primer mix - for L1, L3, L5, L7, N5
                                          5.5 rxn
H2O                              35.6   195.8
Template (ligation product)      10.0    -
Primer mix (f+r) (10uM)           4.0    22.0
SYBR 50X                          0.4     2.2
2X iProof enzyme mix             50.0   275.0
---------------------------------------------
                                100.0   495.0/5.5=90 --- 10uL template
Master mix - Alice's primers - for L2, L4, L6, L8, N6
                                          5.5 rxn
H2O                              31.6   173.8
Template (ligation product)      10.0    -
Primer f(10uM)                    4.0    22.0
Primer r(10uM)                    4.0    22.0
SYBR 50X                          0.4     2.2
2X iProof enzyme mix             50.0   275.0
---------------------------------------------
                                100.0   495.0/5.5=90 --- 10uL template
  • Using the new program modifed based on Dr.Zhang's program (posted on 9-21-'09)
    • Use 5 cycles for the first annealing temp(64).
    • 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
  • Always terminate/pause the reaction at annealing step (72C).
  • Usually the reaction will be close to saturation status around 7~8 cycles. Press the skip buttom to jump to final annealing step when it's closing to saturation stage.
  • Check the qPCR product (3uL) on TBE gel to evaluate the library results.
File:Sam102709-TBE Solexa lib qPCR results-2.jpg
  • The library sizes are as expected. The missed L6 sample was shown on the right size on N6 sample (The additional cutting for L6 sample)
    • 270bp + Y-adaptor = 370bp; 130bp + Y-adaptor = 230bp
  • The primer mix I made showed a stronger primer remains (~25bp) on the gel.
  • Little smear were shown on L4 and L8 samples which may be caused by a over amplification.

Take home messages[edit]

  • Elute DNA in EB after QIAquick washing is ok. (Not need to be eluted in H2O)
  • Y-adaptor I made is working.
  • All of the enzymatic reactions should be set up on cold block
  • No inactivation step is needed in each reaction. Direct using QIAquick washing to terminate the enzymatic reaction.
  • For the short insert (<200bp), the migration of ligation product on PAGE gel might be biased by the Y-adaptor's sturcture. Therefore a denatured PAGE (TBU) gel is recommanded for the size selection of ligation products. Using 5-well comb TBU is recommanded in order to prevent sample over-loading problem.
  • Sample concentration is a key factor too. Use whole amount of product for any following step. The starting amount could be as low as 400ng if there is no size selection after sample shearing.