Sam:LabNotes/Microbiome-new/2011-4-27: Difference between revisions

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*The AMpure beads can remove unwanted size under 100bp, but there still are over-size(>500bp) molecular in the library c5.
*The AMpure beads can remove unwanted size under 100bp, but there still are over-size(>500bp) molecular in the library c5.


==2nd size selection(05-01-'11)==
==2nd size selection (05-01-'11)==
*The purified library is ~20uL. Split sample in two 10uL volume and run 10uL sample/well on a 5-well TBE-PAGE
*The purified library is ~20uL. Split sample in two 10uL volume and run 10uL sample/well on a 5-well TBE-PAGE
**Sample well: 10uL H2O + 10uL 6X loading dye + 10uL DNA sample
**Sample well: 10uL H2O + 10uL 6X loading dye + 10uL DNA sample

Revision as of 23:37, 1 May 2011

Mouse gut bacteria SAG ("c5") preparation - Library amplification from ligation products

Objective

Illumina library amplification references

Procedures

  • Prepare ice box. Clean the PCR hood. Set up realtime PCR program.
  • UV the following tubes:
  1. PCR tube strip x1(Bio-rad) cap x1(Bio-rad)
  2. 0.5mL tube x1 (for master mix)
  3. Cold block
  • Centrifuge the ligation products. 2min, 10000rpm, RT. Leave on ice.
  • Thaw the primers:N2-indexed-Fs(100uM) and N2-indexed-10R(100uM). Diluted them into 10uM (5uL stock sol. + 45uL H2O)
  • Thaw 2X Phusion HF enzyme mix
  • Setup the qPCR program.
  • Lib-amp PCR(100uL): (modified from Dr.Zhang recipe)
                            1rxn   2.5 rxn
H2O                         34     85.0   uL
Adapter ligated DNA         10       -    uL
PCR_F(10uM)                  2      5.0   uL
PCR_R.N2 Index(10uM)         2       -    uL --- using N2-indexed-primer "10"
20X EVA Green                2      5.0   uL
2X Phusion HF MM            50    125.0   uL
                          ------------------
                           100    220.0/2.5=88 --- template 10uL, Index primer(R) 2uL
  • NOTE1: I also amplify another library c6 to balance with c5.
  • NOTE2: N2-index-(10)R primer was used for both C5 and C6 libraries.
  • The reactions tubes were pre-cut before incubation.
    • Using new two-step PCR program. qPCR was performed using Eppendorf realplex machine:
 98C 30sec -> (98C 10sec -> 65C 20sec -> 72C 20sec) x 8 cycles -> (98C 10sec -> 72C 20sec) x 10 cycles -> 72C 3min -> hold 15C. 
  • I will expect the stop the reactions around 15th~16th cycle or the monitored dif. of Flouresscensce reach 14000. Depends on which one reach first.
  • Aliquote 3uL of amplified library for TBE-PAGE validation later.
  • Use AMpure beads(1V, 100uL) to purify the rest of libraries. Elute in 20uL H2O. Aliquote 1uL of purified library for TBE-PAGE validation.
  • For TBE-PAGE validation. Different sample amount were loaded since purified sample is much more concentrated, and I don't want to waste it on gel loading.
    • Un-purified samples(3uL) and purified sample(1uL).
  • TBE-PAGE
    • Unpurified sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer
    • Puriried samplewell  : 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
    • Ladder well  : 1uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
    • Gel was run at 200V for 30min.
    • Use 3uL SYBR-gold with regular stainig(5~10min) with foil protection

Results

File:042811-amp lib c5 and c6.png

  • The AMpure beads can remove unwanted size under 100bp, but there still are over-size(>500bp) molecular in the library c5.

2nd size selection (05-01-'11)

  • The purified library is ~20uL. Split sample in two 10uL volume and run 10uL sample/well on a 5-well TBE-PAGE
    • Sample well: 10uL H2O + 10uL 6X loading dye + 10uL DNA sample
    • Ladder well: 20uL H2O + 10uL 6X loading dye + 0.5uL Low mass ladder/1kb plus ladder
    • Run at 200V for 30min. Long staining (15min with foil wrap protection).
    • Band size between 350~450bp was cut for c5 lib samples in both wells.
  • Shred the gel slice using pole-punctured 0.5mL non-sticky tubes. Centrifuge at 14,000rpm for 3min.
  • Incubate the gel slice mixture with 500uL clean 1X TE at 37C for 2hr on the vortexer.
  • Pellet the gel suspension at 14,000rpm for 1.5min at RT.
  • Transfer the supernatant onto Nanosap filter. Centrifuge at 14,000rpm for 1.5min at RT.
    • NOTE: I got ~350uL supernatant from each of samples.
  • Transfer the filtrate into clean 1.5-mL non-sticky tubes.
  • EtOH precipitation: 2.5V 100%EtOH (875uL), 0.1V 3M NaOAc(35uL), 1.5uL Glycoblue. Mix well by inverting and incubate at -80C for 25min
  • Centrifuge at 10,000xg 4C for 25min. Wash pellet with 750uL 75% EtOH, centrifuge at 10,000xg 4C for 5min. Air dry 5min. Elute the pellet in 15uL H2O. Pool eluted DNA together from two samples => 15X2 = 30uL.
  • Labeled the library as "HC-MDA-Mouse-2h-C5-Mar02"