Sam:LabNotes/Microbiome-new/2010-3-2
Jump to navigation
Jump to search
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
- E.coli internal marker (IM) (200bp): 43.6ng/uL
- 1/10 diluted E.coli internal marker (IM) (200bp): 4.36ng/uL
- DNAse digested-cleaned (D-C) tRNA 109 ng/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.
End-reparing & 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
- 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 0.8 70.2 DNA 20.0 - 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
- The amplification was stopped at the 7th 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:Sam022210-2nd PCR lib.JPG L1 L2 L3 L4 L5 L6 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
- The the Dephosphorylated carriers are still ligated with adaptor in this tests.
- Sol1: Adjust the dephosphorylation conditions.
- Sol2: Add heat inactivation stet right after End-reparing reaction.
- The smear (unspecific amplification) is weaker in those samples with lambda DNA carriers. Perhaps the Y-adaptor were comsumed more in those samples.
- I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.
- In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.