Sam:LabNotes/Human/2009-12-17: Difference between revisions

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'''NOTE:
'''NOTE:
*For the qPCR amplification. I also tried the newly synthesized primer. Although the templates were succesuflly amplified,
*For the qPCR amplification. I also tried the newly synthesized primer. 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 amplification curves were not consistent with the TBE-PAGE results. I am wondering if my qPCR didn't perform well on final
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.
**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.
**I will need to try the qPCR amplification on those newly synthesized primer again.

Revision as of 08:19, 25 December 2009

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.

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.
  • Stored the rest of unpurified samples in -20C.

NOTE:

  • For the qPCR amplification. I also tried the newly synthesized primer. 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.