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

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
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.