Sam:LabNotes/Human/2009-10-29: Difference between revisions

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>Sam Chiang
>Sam Chiang
Line 42: Line 42:
  10X buf.      4      10 (10X Knenow buf.)
  10X buf.      4      10 (10X Knenow buf.)
  1mM dATP      6      15
  1mM dATP      6      15
  Klenow enzyme         2      5
  Klenow enzyme 2      5
  ------------------------------------------
  ------------------------------------------
               40      30/2.5=12 - DNA 30uL
               40      30/2.5=12 - DNA 30uL

Revision as of 06:21, 1 November 2009

Solexa sequencing library construction - real samples (human chro MDA product)

Background

  • Sample information
    • Sample No.10: 1/12.5 (200chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL)
    • Sample No.12: 1/25 (100chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL)
  • Only 10uL of each sample were used for this construction (save another 10uL sample as backup).

TBE-PAGE size selection

  • TBE-PAGE condition: 5-well TBU. Run at 200V, 30min
    • Sample: 10uL sample + 5uL H2O + 5uL 6X loading dye
    • Ladder: 0.5uL 25bp ladder + 15uL H2O + 5uL 6X loading dye
    • Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
    • Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
  • Cut the size 250+/-50bp (200bp~300bp)
File:Sam102909-TBE-size selection.JPG File:Sam102909-TBE-size selection-post cutting.JPG
          Left: Pre-cutting                              Right: Post-cutting

Blunting

  • End-it Kit (EPICENTRE)
    • Set reactions on cold block
                      1rxn     2.5rxn
DNA                   28.0      -
10X blunting buf.      4.0     10.0
dNTP mix           4.0     10.0
Blunting Enzyme        4.0     10.0
------------------------------------
                      40.0     30.0   30/2.5=12 --- DNA 28uL
  • 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.

A-tailing

  • Exo-Minus Klenow DNA polymerase(EPICENTRE)
    • Set reactions on cold block
  • Prepare the fresh dilution of 100mM dATP to 1mM dATP
              1rxn    2.5rxns(EPI)
DNA           30        -
10X buf.       4      10 (10X Knenow buf.)
1mM dATP       6      15
Klenow enzyme  2       5
------------------------------------------
              40      30/2.5=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 only 25% DNA left after all of previous steps. 360ng*25% =90ng
    • DNA before ligation: 90ng of 250b
    • 90/(660x250)x10^6/1000 = 0.54uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 5.4uM ~ 27uM Y-adaptor needed.
    • I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule.
                              2.5rxn 
 A-tailed DNA           11     -
 2X rapid liagas buf.   15    37.5
 20uM Y-adaptor          2     5.0
 T4 ligase               2     5.0
 ---------------------------------
                        30    47.5/2.5 = 19uL --- DNA 11 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.

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 H2O + 10uL 2X TBU-loading dye
    • Heat denaturing: 75C 15min -> 1min on cool block
    • Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
    • 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

  • 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

  • Only use helf 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

  • 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.