Sam:LabNotes/Microbiome-new/2011-4-27
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Mouse gut bacteria SAG ("c5") preparation
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 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
- 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 to purify the rest of libraries. Elute in 20uL H2O. Aliquote 1uL of purified library for TBE-PAGE validation.
Results
- I also amplify another library c6 to balance with c5.
- 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
File:042811-amp lib c5 and c6.png
Next
- Perform 2nd size selection to narrow down the size of library.