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== | ||
Line 13: | Line 13: | ||
*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]
- 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[edit]
- Ligation product amplification with N2-indexed primer
- AMpure bead purification
- 2nd size-selection
Procedures[edit]
- 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[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"