Sam:LabNotes/Microbiome-new/2010-3-2
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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
Sample name B1 B2 B3 B4 B5 B6 B7 B8 200ng IM 50ng IM 10ng IM 50ng IM 10ng IM 0ng IM 0ng IM 0ng IM 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA 500ng tRNA 500ng tRNA --------------------------------------------------------------------------------------------------------------------------------------------- E.coli IM (43.6ng/uL) 4.59 1.15 - 1.15 - - - - 1/10 E.coli IM (4.36ng/uL0 - - 2.29 - 2.29 - - - D-C-tRNA (78ng/uL) 3.85 5.77 6.28 5.77 6.28 6.41 6.41 6.41 H2O 91.56 93.08 91.43 93.08 91.43 93.59 93.59 93.59 --------------------------------------------------------------------------------------------------------------------------------------------- 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 uL NOTE: reactions B1, B2 and B3 will be added with De-P-carrier. Reactions B4 - B8 won't.
- Purify the DAN mixture with EtOH precipitation
- mimic the real precedures to remove the enzyme and buffers from WGA reactions
- Each sample (PCR product) has 100uL volume. Air dry the pellet. Dissolve the pellet in 34uL H2O.