Sam:LabNotes/Microbiome-new/2010-11-14
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Illumina libraries pooling(short and long amplified E.coli SAGs -3rd test 11-07-'10)[edit]
Background[edit]
- Eight librareis from short and long amplified E.coli SAGs (3rd test, 11/07/2010) were prepared. Eight libraries were amplified in two batches (2x8 =16 libraries).
- I attemp to pool the libraris and do another size selection to get a narrower region for sequencing.
- In order to do size selection, I need to decrease the sample volume. Decreasing sample volume could be achieved through purification with small elution volume.
- I still have to keep individual samples for the possibility of pooling with different combination in the future, so purification sample individually is a better strategy.
1. Perform 1V AMpure bead purifcation of each individual library and elute with 20uL pre-warmed H2O. 2. Quantify the purified samples by Nanodrop. 3. Pool the samples with equal amount (control total volume within 60~80uL). 4. Perform size selection using 5-well PAGE and 1kb ladder. 5. Recover DNA from the cut gel slice.
- The pooled library will be evaluated again using cloning Sanger sequncing.
Procedures[edit]
AMpure beads purification (multi-channel pipettor)[edit]
Set up: UV-ed Solution Basin - for 70% EtOH (6mL) UV-ed 8-well PCR tube strip (high-profile, Biopioner) UV-ed 8-well PCR tube strip w/ individual cap (high-profile, Eppendorf) -> for collecting purified products
- Load 100uL libraries into PCR tubes.
- Load 1V AMpure beads (100uL). Mix by repeat pipetting 20 times(200uL, multi-channel pipettor). Incubate at RT for 5min.
- Relocate onto magnetic board. Incubate at RT for 2min. Remove the supernatant.
- Add 200uL 70% EtOH into the PCR tubes(no pipetting). Incubate at RT for 1min. Remove the supernatant.
- Repeat the EtOH wash again.
- Use 10p tips to remove the remained EtOH.
- Air dry the the EtOH for ONLY 2min. During the air-drying, relocate PCR tubes to 96-well rack.
- Add 20uL pre-warmed RT-PCR grade H2O. Mix by repeat pipetting 20times(WASH DOWN AMPURE BEADS BY PIPETTING AT DIFFERET DIRECTION). Incubate at RT for 5min.
- Relocate PCR tubes on the magnetic board. Incubate at RT for 2min.
- Carefully transfer the supernatant(eluted DNA) into a fresh PCR tube strip(THE REMAINED MAGNETIC BEADS WILL AFFECT NANODROP READINGS).
Results[edit]
- I did AMpure bead purification for two batches of libraries(1-1~8-1; 1-2~8-2).
Nanodrop quantification: batch 1 File:Sam111310-nanodrop purified lib.jpg Nanodrop quantification: batch 2 File:Sam111410-nanodrop purified lib.jpg
PAGE-gel quantification of purified libraries[edit]
- I noticed that Nanodrop reading may have bias on quantification due to the primer dimmer or amplification background not removed by AMpure beads.
- The concentration difference between short and long amplified libraries should be more than 2-fold as shown on PAGE-gel. http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-07_exp1
- I decided to run the PAGE gel to confirm this hypothesis and also to quantify library concentration.
- I only run PAGE-gel on 1st batch of purified libraries(1-1~8-1)
File:Sam111410-page-semi-quant.jpg File:Sam111410-page-semi-quant-cal.jpg
File:Sam111410-semi-quant.jpg Estimated conc.(ng/uL) Amount for 20ng(uL) 1 - 1 1.36 14.68 uL 2 - 1 0.92 21.83 uL 3 - 1 36.97 0.54 uL 4 - 1 34.87 0.57 uL 5 - 1 8.24 2.43 uL 6 - 1 10.80 1.85 uL 7 - 1 35.42 0.56 uL 8 - 1 28.09 0.71 uL 43.19 uL total
- The estimation by PAGE-gel is quite different from that by Nanodrop. I tend to believe that PAGE-gel estimation is more accurate. The libraries will be pooled by equal amount of 20ng libraries.