Sam:LabNotes/Microbiome-new/2009-8-11: Difference between revisions
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*When the band reach reference line, pause the electrophoresis and fill up the H2O in collecting wells. '''Note: Fill up with H2O using 20p pipetor, but don't let the h2O overflow'''. | *When the band reach reference line, pause the electrophoresis and fill up the H2O in collecting wells. '''Note: Fill up with H2O using 20p pipetor, but don't let the h2O overflow'''. | ||
*Cover the amber filter and continue the gel running. Collect the samples precisely when it reachs the collecting well. Only collect 2uL. | *Cover the amber filter and continue the gel running. Collect the samples precisely when it reachs the collecting well. Only need to collect 2uL from target band. | ||
*The collected DNA (2uL) was diluted in 1/100 when it was resuspended in 198uL H2O. The diluted DNA now could be used for 2nd PCR amplification and validation. | *The collected DNA (2uL) was diluted in 1/100 when it was resuspended in 198uL H2O. The diluted DNA now could be used for 2nd PCR amplification and validation. |
Revision as of 00:54, 7 August 2009
E-gel size selection for mouse microbiome PCR product
Background
- Unspecific PCR amplicon leads to sequencing failure in previous Genewiz results.
- Review all of previous candidates PCR amplicons. Perform E-gel size selection for band purification.
E-gel 2% agarose gel size selection
- Prepare the clean PCR tubes for collection. For 1/100 dilution,add 198 uL Nuclease-free H2O into the PCR tubes.
- Set up E-gel machine. Open a new gel and remove the comb. Assemble the gel into the machine.
- Load 20uL PCR products into each of the upper row wells. Load 6uL diluted ladder in the "M"-well. filled up the empty well with 20uL diH2O (low quality H2O is fine)
- Fill up 20uL of nuclease-free H2O in the (lower row) collecting wells and 6~10 uL H2O for the "M" well.
- Choose program 8 (Size selecton 2%). For 500bp dsDNA, it takes about 10 min to reach reference line. Cover the amber filter and press the "go" button to run. The gel could be checked realtime when the flourescent light was turned on. Note: Make sure the amber filter was covered before turn on the flouresecnt background!!!
- It is recommanded to record the relative bands position for each well, which helps a lot for targeting the correct band.
- When the band reach reference line, pause the electrophoresis and fill up the H2O in collecting wells. Note: Fill up with H2O using 20p pipetor, but don't let the h2O overflow.
- Cover the amber filter and continue the gel running. Collect the samples precisely when it reachs the collecting well. Only need to collect 2uL from target band.
- The collected DNA (2uL) was diluted in 1/100 when it was resuspended in 198uL H2O. The diluted DNA now could be used for 2nd PCR amplification and validation.