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='''Illumina libraries pooling(short and long amplified E.coli SAGs -3rd test 11-07-'10)'''= ==Background== *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). **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-11-07_exp1 *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== ===AMpure beads purification (multi-channel pipettor)=== 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=== *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|800px]] Nanodrop quantification: batch 2 [[File:sam111410-nanodrop purified lib.jpg|800px]] ===PAGE-gel quantification of purified libraries=== *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|450px]] [[File:sam111410-page-semi-quant-cal.jpg|400px]] [[File:sam111410-semi-quant.jpg|500px]] 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'''.
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