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

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>Sam Chiang
>Sam Chiang
 
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Line 24: Line 24:
  DNA                  28.0      -
  DNA                  28.0      -
  10X blunting buf.      4.0    10.0
  10X blunting buf.      4.0    10.0
  dNTP mix           4.0    10.0
  dNTP mix               4.0    10.0
  Blunting Enzyme        4.0    10.0
  Blunting Enzyme        4.0    10.0
  ------------------------------------
  ------------------------------------
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
Line 55: Line 55:
*Adaptor:Insert calculation
*Adaptor:Insert calculation
**Assume that only 25% DNA left after all of previous steps. 360ng*25% =90ng
**Assume that only 25% DNA left after all of previous steps. 360ng*25% =90ng
**DNA before ligation: 90ng of 250b
**DNA before ligation: 90ng of 250bp
**90/(660x250)x10^6/1000 = 0.54uM --- Insert.  Using 10:1~50:1 (Adaptor: Insert)will be 5.4uM ~ 27uM Y-adaptor needed.
**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.
**I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule.
Line 78: Line 78:
**Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
**Stained with 3uL SYBR-Gold in 0.5X TBE for 5min


  [[Image:sam102609-TBU-4th test.jpg|450px]] [[Image:sam102609-TBU-4th test -post cut.jpg|450px]]
  [[Image:sam103009-TBU.jpg|400px]] [[Image:sam103009-TBU-post cutting.jpg|380px]]
  Left: Pre-cutting                                        Right: Post-cutting
  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==
==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.
*Gel-cut slice was sheared by centrifuging at 14000 rpm at RT, 3min using a 22G needle punched 0.5mL tube.
*Transfer the sliced gel in 0.5mL tubes. Centrifuge at 14000 rpm at RT for 3min.
*Sheared gel was soaked in 400uL clean 1X TE and incubate at 37C for 2hr on the shaker. shaking at lowest speed.
*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.
*Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant on a Nanosep column filter.  
*Incubate the gel-TE mixture at 37C for 1hr on the shaker (shaking at lowest speed)
*Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through.
*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.
*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. Incubate at -80C for 25min (or longer)
**Gentaly invert the tube 20times. Incubate at -80C for 25min (or longer)
**Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 25min. Remove the supernatant.
**Centrifuge at 4C, 10,000xg for 25min. Remove the supernatant(pour + 20p tips). Wash the pellet with 75% EtOH. Gentaly invert the tube 20times.
**Wash the pellet with 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant.
**Centrifuge at 4C, 10,000xg for 5min. Remove the supernatant(pour + 20p tips). Air dry the pellet in PCR hood (~10min).
**Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
**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.
[[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 02:31, 17 December 2009

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

Background[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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 250bp
    • 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[edit]

  • 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:Sam103009-TBU.jpg File:Sam103009-TBU-post cutting.jpg
Left: Pre-cutting                                        Right: Post-cutting

Size selected DNA recovery from TBU-PAGE[edit]

  • 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 for 2hr on the shaker. shaking at lowest speed.
  • Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant on 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.