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.
  • 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.