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
- Ecoli DNA (EDNA)
- Single-bacterium WGA amplicon P11-1, P12-1 (from 01-27-'10 exp.)
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.
End-repairing & Phosphorylation
- End-it Kit (EPICENTRE)
1rxn 8.5rxn DNA+H2O 34.0 - 10X buf. 5.0 42.5 2.5mM dNTP 5.0 42.5 10mM ATP 5.0 42.5 Enzyme 1.0 8.5 ------------------------------------ 50.0 136.0 136/8.5=16
- Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
- Adding De-P-Carrier into the DNA mixture during QIAquick washing.
- Concentration of De-P-Carrier 1ug/30uL = 33.3ng/uL
- S30: 450ng for B4 reaction: 450/33.3=13.5uL
- S30: 490ng for B5 reaction: 490/33.3=14.7uL
- S30 :500ng for B6 reaction: 500/33.3=15.0uL
- S60 :500ng for B7 reaction: 500/33.3=15.0uL
- A60 :500ng for B8 reaction: 500/33.3=15.0uL
- Elute DNA in 28uL EB
A-tailing
- Exo-minus klenow DNA pol. kit (Epicentre)
1rxn 8.5rxn DNA+H2O 28.0 - 10X buf. 4.0 34.0 1mM dATP 6.0 51.0 Enzyme 2.0 17.0 ------------------------------------ 40.0 102.0 102/8.5=12
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
- QIAquick purification. Eluted in 20uL EB.
ligation
- T4 ligation kit (Enzymatic)
1rxn 8.5rxn DNA+H2O 11.0 - 2X rapid buf. 15.0 127.5 20uM Y-adaptor 2.0 17.0 T4 ligase 2.0 17.0 ------------------------------------ 30.0 161.5/8.5=19
- Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
- QIAquick purification. Eluted in 20uL EB.
Size selection
- Only size select the first six reactions (reaction B7 and B8 are two extra negative controls).
- 20uL PCR product samples were run on 5-well PAGE (200V, 30min).
- Sample well: 20uL sample + 10uL 6X loading dye + 10uL 0.5X TBE buffer
- Ladder well: 0.5uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer
File:Sam022810- B1 to B4 pre-cut.jpg File:Sam022810- B1 to B4 post-cut.jpg File:Sam022810- B5 to B8 pre-cut.jpg File:Sam022810- B5 to B8 post-cut.jpg The dual bands of ligation reactions suggested that some DNA were only ligated with one end but not both ends
- DNA was recovered from the gel slice
- During the 37C incubation step (1X TE and homogenized gel slides), additional tRNA carriers were added into each reaction to protect sample DNA.
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 --------------------------------------------------------------------------------------------------------------------------------------------- D-C-tRNA(78ng/uL) 3.85 5.77 6.28 5.77 6.28 6.41 6.41 6.41 uL
- Purify the DAN with Nanosap filter and EtOH precipitation.
- Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
Libraray amplification by qPCR
- 2X Fusion mastermix (Enzymatic)
1rxn 6.5rxn H2O 10.8 70.2 DNA 10.0 - PCR_f (10uM) 2.0 13.0 PCR_r (10uM) 2.0 13.0 SYBR 50X 0.2 1.3 2X Fusion enzyme 25.0 162.5 ------------------------------------ 50.0 195.0/6.5=30
- Only use 10uL (out of 20uL) template.
- The amplification was stopped at the 8th cycle and 12th cycle.
TBE-PAGE validation
- 3uL PCR products were run on 10-well PAGE (200V, 30min).
- Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer
- Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
File:Sam030210-PCR amplified lib - 8th cycle.JPG File:Sam030210-PCR amplified lib - 12th cycle.JPG The library from B5 didn't showed up in 8th cycle due to the insufficinet amplification. B1 B2 B3 B4 B5 B6 Input DNA 200ng 50ng 10ng 50ng 10ng 0ng tRNA 300ng 450ng 490ng 450ng 490ng 500ng De-P-Carrier 450ng 490ng 500ng Expected size: Nucleotides Prior-ligation Post-ligation -------------------------------------------------- tRNA N/A N/A Sample (E.coli) 200bp ~300bp Carrier (Lambda) 100bp ~200bp
Discussion
- Regardless which enzyme or condition was used, all dephosphorylated carriers are still ligated with adaptor in this test.
- Even I did the inactivation step (70C 10min) following the End-repairing step, the situation was not improved.
- B4-B6: De-P-Carrier prepared by SAP, 30min.
- B5: De-P-Carrier prepared by SAP, 60min.
- B6: De-P-Carrier prepared by ACP, 60min.
- Possible reasons of the appearance of adaptor-ligated carrier:
- The Dephosphorylation is not complete.
- The De-P-Carriere was rephosphorylated after End-repairing.
- The Phosphorylation "on templates (insert)" is not playing the critical roll for ligation.
- The early size selection method is able to construct the library with only 10ng DNA (for both with or without carrier conditions).
- The early size selection method can generate libraries with much less unspecific amplification.