Sam:LabNotes/Human/2009-12-17

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Revision as of 08:40, 4 January 2010 by >Sam Chiang (→‎Product purification)
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Solexa sequencing library construction - human single cell MDA amplicons (1,10,1+10hr incubation)

Background

  • The MDA amplicons were EtOH purified and followed by NlaIII digestion (12-16-'09). Total voulmn is 20uL.
  • Due to the loading capacity of TBE gel. Only 10uL was used for library construction. The starting amounts for each samples are:
    • ~0.2 (ng/uL) * 8uL / 20uL x 10uL = 0.8 ng - for 1hr-incubation samples (1-1, 1-2)
    • ~50 (ng/uL) * 8uL / 20uL x 10uL = 200 ng - for 10 and 10+1 hr-incubation samples (10-1, 10-2, 11-1, 11-2)

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
    • Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
  • Cut the size 250+/-50bp (200bp~300bp)
File:Sam121609-precut-sample 1-2-3.JPG File:Sam121609-postcut-sample 1-2-3.JPG

File:Sam121609-precut-sample 4-5-6.JPG      File:Sam121609-postcut-sample 4-5-6-postcut.JPG
          Left: Pre-cutting                              Right: Post-cutting

Sample annotation: 
#1-1(1hr-T3), #1-2(1hr-T6), #10-1(10hr T2), #10-2(10hr-T5), #11-1(1+10hr T3), #11-2(1+10hr T6)

NOTE: The starting amount of DNA for sample #1-1 and #1-2 are too few to been seen on PAGE-TBE gel.

  • I only perform the DNA recovery for 10-1,10-2, 11-1 and 11-2 samples.

Size selected DNA recovery from TBE-PAGE

  • 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 1hr on the shaker. shaking at lowest speed.
  • Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant (~300uL)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.

Blunting

  • End-it Kit (EPICENTRE)
    • Set reactions on cold block
                      1rxn     4.5rxn
H2O                    8.0      - 
DNA                   20.0      -
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
  
Add 1uL of DNA pol.I (E.coli) for nicks filling on each reaction - (These are NlaIII digested samples)
  • 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 Pre-wrmed 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    4.5rxns(EPI)
DNA           30       -
10X buf.       4       18 (10X Knenow buf.)
1mM dATP       6       27
Klenow enzyme  2        9
------------------------------------------
              40       54/4.5=12 - DNA 30uL
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min at Thermocycler.
    • 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. 200ng*25% =50ng
    • DNA before ligation: 90ng of 250bp
    • 50/(660x250)x10^6/1000 = 0.303uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 3.03uM ~ 15.15uM Y-adaptor needed.
    • I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule.
                              4.5rxn 
 A-tailed DNA           11     -
 2X rapid liagas buf.   15    67.5
 20uM Y-adaptor          2     9.0
 T4 ligase               2     9.0
 ---------------------------------
                        30    85.5/4.5 = 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:Sam121809-ligation size selection -precut.jpg File:Sam121809-ligation size selection -postcut.jpg
Left: Pre-cutting                                        Right: Post-cutting

Size selected DNA recovery from TBU-PAGE

  • 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 1hr 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.

qPCR amplification

  • Only use half of amount(10uL) of ligation product for qPCR amplification.
  • Solexa lib. templates:
    • Left to right: 10-1, 10-2, 11-1, 11-2
  • Primers: From Dr.Zhang (Yellow-cap)
    • Working solution: 10uM
Master mix
                                          4.5 rxn
H2O                                31.6   142.2
Template                           10.0     -
PCR_F (10uM)                        4.0    18.0
PCR_R (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) - step5.
    • Use 16 cycles for the second annealing temp(72C) - step9.
  • Immediately transfer 50uL of reaction into a fresh PCR tube for an extra cycle amplification in Chromo4.
  • Terminate/pause the reaction at annealing step when it's closing to saturation stage.
File:Sam122109-libamp-step5.jpg  File:Sam122109-libamp-step6 and above.jpg
The reaction were terminated at: 10-2, 11-1(8th cycle), 10-1(9th cycle), 11-2(10th cycle)

TBE-PAGE validation

    • Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE
    • Ladder: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE
    • 10-well TBE gel. Run at 200V for 30min.
File:Sam122209-qPCR amplib.jpg
The sample 10-1 and 11-2 seem a little bit over amplified.
The expected size is accurate on 400bp (300bp sample + 100bp adaptor)

Product purification

  • Purify the amplified library with QIAquick column.
    • Take 20uL sample and eluted with 15uL EB.
  • Gave the purified libraries to Alice for Solexa sequencing.
 Lib. name    Sample 
 sam003       10-1
 sam004       10-2
 sam005       11-1
 sam006       11-2
  • Stored the rest of unpurified samples in -20C.

NOTE:

  • For the qPCR amplification. I also tried the newly synthesized primer(PCR_F and PCR-R). Although the templates were succesuflly amplified, the amplification curves were not consistent with the TBE-PAGE results.
  • I am wondering if my qPCR didn't perform well on the final vortexing and spining step. There may have some reaction sticked to the tube cap.
    • The current results were completed using the original qPCR primer (From Dr. Zhang) by a repeat qPCR reaction.
    • I will need to try the qPCR amplification on those newly synthesized primer again.