Sam:LabNotes/Microbiome-new/2010-3-2: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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==Background== | ==Background== | ||
*Both of following protocols were working for low-input DNA library construction: | *Both of following protocols were working for low-input DNA library construction: | ||
**Size selection prior PCR amplification (with/without De-P-Carrier) | **Size selection '''prior PCR amplification''' (with/without De-P-Carrier) | ||
**Size selection after PCR amplification (with/without De-P-Carrier) | **Size selection '''after PCR amplification''' (with/without De-P-Carrier) | ||
*I decided to use the method with size selection prior PCR amplification, but without adding De-P-Carriers. | *I decided to use the method with size selection prior PCR amplification, but without adding De-P-Carriers. | ||
*This experiment also includes the tests of different amount of positive control using sheared DNA. | *This experiment also includes the tests of different amount of positive control using sheared DNA. | ||
==Procedures== | ==Procedures== |
Revision as of 06:09, 3 March 2010
Single-bacterium DNA library construction - test 1
Background
- Both of following protocols were working for low-input DNA library construction:
- Size selection prior PCR amplification (with/without De-P-Carrier)
- Size selection after PCR amplification (with/without De-P-Carrier)
- I decided to use the method with size selection prior PCR amplification, but without adding De-P-Carriers.
- This experiment also includes the tests of different amount of positive control using sheared DNA.
Procedures
- Prepare the following samples:
- DNAse digested-cleaned (D-C) tRNA 83ng/uL
Sample name C1 C2 C3 C4 C5 C6 200ng EDNA 50ng EDNA 10ng EDNA 50ng P11-1 50ng P12-1 50ng IM 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 450ng tRNA 450ng tRNA -------------------------------------------------------------------------------------------------------------------- Sheared DNA 50.00 50.00 50.00 50.00 50.00 - E.coli IM (43.6ng/uL) - - - - - 1.15 D-C-tRNA (83ng/uL) 3.60 5.42 5.90 5.42 5.42 5.42 H2O 46.40 44.58 44.10 44.58 44.58 93.43 --------------------------------------------------------------------------------------------------------------------- 100.00 100.00 100.00 100.00 100.00 100.00 uL
- Purify the DAN mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O.