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

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>Sam Chiang
>Sam Chiang
 
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='''Mouse gut bacteria SAG ("c5")preparation'''=
='''Mouse gut bacteria SAG ("c5") preparation - Library amplification from ligation products'''=


==Objective==
==Objective==
*I prepared two Illumina libraries using limited MDA (2hr incubation) strategy, but only one of the samples was sequenced.
*I prepared two Illumina libraries using limited MDA (2hr incubation) strategy, but only one of the samples was sequenced.
**
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-2
 
*I still have the ligation product left from this sample. I will amplify it using N2-index primer and give it to Alan for sequencing.


==Illumina library amplification references==
==Illumina library amplification references==
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*2nd size-selection
*2nd size-selection
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-15
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-15
==Procedures==
*Prepare ice box. Clean the PCR hood. Set up realtime PCR program.
*UV the following tubes:
#PCR tube strip x1(Bio-rad)  cap x1(Bio-rad)
#0.5mL tube x1 (for master mix)
#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.
**In AMpure bead manual, 1.8V beads are recommended.
*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|350px]]
*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.
[[File:050111-c5 library precut.png|350px]]  [[File:050111-c5 library post-cut.png|370px]]
*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'''"

Latest revision as of 22:27, 9 July 2011

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

Objective[edit]

Illumina library amplification references[edit]

Procedures[edit]

  • 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.
    • In AMpure bead manual, 1.8V beads are recommended.
  • 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[edit]

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)[edit]

  • 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.
File:050111-c5 library precut.png  File:050111-c5 library post-cut.png
  • 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"