Sam:LabNotes/Microbiome-new/2011-4-27: Difference between revisions
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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 | |||
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. | |||
5.Aliquote 3uL of amplified library for TBE-PAGE validation later. | |||
6.Use AMpure beads to purify the rest of libraries. Elute in 20uL H2O. Aliquote 1uL of purified library for TBE-PAGE validation. |
Revision as of 02:19, 29 April 2011
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.
5.Aliquote 3uL of amplified library for TBE-PAGE validation later. 6.Use AMpure beads to purify the rest of libraries. Elute in 20uL H2O. Aliquote 1uL of purified library for TBE-PAGE validation.