Sam:LabNotes/Microbiome-new/2011-4-27: Difference between revisions
Jump to navigation
Jump to search
>Sam Chiang |
>Sam Chiang |
||
Line 68: | Line 68: | ||
**Run at 200V for 30min. Long staining (15min with foil wrap protection). | **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. | **Band size between 350~450bp was cut for c5 lib samples in both wells. | ||
[[File:050111-c5 library precut.png| | [[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. | *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. | *Incubate the gel slice mixture with 500uL clean 1X TE at 37C for 2hr on the vortexer. |
Revision as of 01:09, 2 May 2011
Mouse gut bacteria SAG ("c5") preparation - Library amplification from ligation products
Objective
- I prepared two Illumina libraries using limited MDA (2hr incubation) strategy, but only one of the samples was sequenced.
- 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
- Ligation product amplification with N2-indexed primer
- AMpure bead purification
- 2nd size-selection
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.
- 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.
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"