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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)(Covaris sheared ~200bp) http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-2-4 **Single-bacterium WGA amplicon P11-1, P12-1 (from 01-27-'10 exp.)(Covaris sheared ~200bp) **http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-1-27 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 Internal Marker(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 DNA mixture with EtOH precipitation. Air dry the pellet. Dissolve the pellet in 34uL H2O. ===End-repairing=== *End-it Kit (EPICENTRE) 1rxn 6.5rxn DNA+H2O 34.0 - 10X buf. 5.0 32.5 2.5mM dNTP 5.0 32.5 10mM ATP 5.0 32.5 Enzyme 1.0 6.5 ------------------------------------ 50.0 104.0 104/6.5=16 *Mix the reaction by vortexing 5sec. Incubate at RT for 45min. *Purify the DNA with QIAquick. Elute DNA in 28uL EB ===A-tailing=== *Exo-minus klenow DNA pol. kit (Epicentre) 1rxn 6.5rxn DNA+H2O 28.0 - 10X buf. 4.0 26.0 1mM dATP 6.0 39.0 Enzyme 2.0 13.0 ------------------------------------ 40.0 78.0 78/6.5=12 *Mix the reaction by vortexing 5sec. Incubate at 37C for 30min. *QIAquick purification. Eluted in 12uL EB. ===Ligation=== *T4 ligation kit (Enzymatic) 1rxn 6.5rxn DNA+H2O 11.0 - 2X rapid buf. 15.0 97.5 20uM Y-adaptor 2.0 13.0 T4 ligase 2.0 13.0 ------------------------------------ 30.0 123.5/6.5=19 *Mix the reaction by vortexing 5sec. Incubate at RT for 15min. *QIAquick purification. Eluted in 20uL EB. ===Size selection=== *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: 1.0uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer [[File:sam030410-C1 C2 C3.jpg|350px]] [[File:sam030410-C1 C2 C3 - post cut.jpg|400px]] [[File:sam030410-C4 C5 C6.jpg|350px]] [[File:sam030410-C4 C5 C6 - post cut.jpg|350px]] *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''' 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 -------------------------------------------------------------------------------------------------------------------- D-C-tRNA (83ng/uL) 3.60 5.42 5.90 5.42 5.42 5.42 uL *Incubate the gel mixture with '''500uL 1X TE at 37C for 2hr'''. *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 Phusion HF enzyme 25.0 162.5 ------------------------------------ 50.0 260.0/6.5=30 *'''Only use 10uL (out of 20uL) template'''. *The amplification was stopped at the 8th cycle and 10th 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:sam030510-lib amp 8th cycle.jpg|320px]] [[File:sam030510-lib amp 10th cycle.JPG|300px]] ==Discussion== *All libraires were working as we expected. *For the C5 sample, '''it is unknown why 10th cycle amplification has less background than 8th cycle amplification'''. I run another gel to check them (results not shown) and results are still the same. *The libraries C4 and C5 (from single cell genome 11-1 and 12-1, respectively) were ready for sequencing. **16S-PCR info: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-1-28 **After discussed with Dr. Zhang, we will prefer to sequence sample from E.coli single cell genome in which we can optimized the protocol of library construction. **We hold the sequencing of the C4 and C5 librery.
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