Sam:LabNotes/Microbiome-new/2010-8-4
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Nextera-library construction on single E.coli MDA amplicons(0727'10 MDA)[edit]
Background[edit]
- Two Ext-MDA amplicons (single E.coli) were found E.coli-positive. http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/Microbiome/2010-8-2.
- The positive amplicons are:
- Well-3(E)-10hr-sam072710, well-4(F)-10hr-sam072710
- I did the QUBIT-HS for both amplcons and their original 90m amplicons.
File:Sam080410 - QUBIT -new.jpg
- Four samples will be made Nextera libraries with barcoded primers.
Sample preparation[edit]
Name DNA(20ng) tRNA carrier(480ng) RT-PCR H2O ---------------------------------------------------- 90m-3 45.8 4.8 49.4 10h-3 2.0 4.8 93.2 90m-4 40.0 4.8 55.2 10h-4 2.3 4.8 92.9
- Only use about 20ng DNA for starting amount (10ng is the total available amount for the sample Sam072710-90m-3)
EtOH purification of MDA amplicons[edit]
- Add 4.8uL tRNA carrier(100ng/uL) into MDA amplicon.
- Add H2O to make total valume to 100uL.
- Add 2.5X volume 100% EtOH (250uL) + 0.1X volume 3M NaOAc (10uL) + 1.5uL Glycoblue -> incubate at -80C for 25min.
- Centrifuge at 10,000xg, 4C for 25min -> resuspend the pellet with 750uL 75% EtOH.
- Centrifuge at 10,000xg, 4C for 5min. Elute the pellet in 15uL RT-PCR grade H2O.
Tagmentation reaction[edit]
- Set up tagmentation reaction on cold block:
1rxn DNA(10ng)+ H2O 15.0 5X Nextera Rxn buf.(LMW) 4.0 Nextera enzyme 1.0 ------------------------------ 20.0 uL
- Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min.
- During the incubation, setup the tube and buffer of Zymo DNA cleaning kit
Zymo DNA purification[edit]
- Pre-load 100uL binding buf. in 1.5mL-tubes. Add 20uL reaction product from last step.
- Briefly vortexing and centrifuging.
- Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube.
- Centrifuge at 10,000xg for 1min. Discard flowthrough.
- Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flow-through.
- Repeat the last step again.
- Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue).
- Add 11uL pre-warmed H2O onto the column, transfer the column onto a 1.5mL-tube. Incubate at RT for 2min.
- Centrifuge at 10,000xg for 1min. The elute DNA is about 10~11uL. Only use 5uL for the library amplification. (Label and store the other 5uL product in -20C)
Library enrichment by PCR amplification[edit]
- In order to use the libraries for cloning sequencing, we have to switch to the Phusion enzyme instead.
- The Phusion enzyme provide blunt end PCR products which is suitable for blunting cloning.
- We also need to add Isotherm (Epicentre) enzyme to repair the gaps left by transposon taggmentation.
- In order to test the new amplification recipe, I only tried two of four samples with barcoded(Index) primer
- 90m-4(IDX1) and 10hr-4(IDX2)
- Set up the qPCR reaction (50uL) on cold block in PCR tubes:
1rxn RT-PCR grade H2O 17.1 DNA lib (from last step) 5.0 2X Phusion master mix 25.0 50X Nextera primier cocktail 1.0 - Illumina-compatible 50X Nextera adaptor 2 1.0 - Using IDX1 and IDX2 50X SYBR I 0.4 Isothermo(Epicentre) 0.5 --------------------------------- 50.0 uL
- Briefly vortexing and centrifuging.
- 65C 3min -> 98C 30sec -> (98C 5sec -> 65C 15sec -> 72C 20sec)x15 cycle -> 72C 3min -> 4C forever
- NOTE1.: This program is adapted from Dr.Zhang's protocol 0805'10. The temperature setting is different from original Nextera protocol.
- NOTE2.: The 65C 3min is for Isothermo enzyme. The 98C 30sec is for Phusion (Hot-start) enzyme.
- Stop the reaction before the amplification reach saturation.
- Sample 90m-4 stopped at 16cycle
- Sample 10h-4 stopped at 8cycle
Ampure bead purification[edit]
- Following standard protocol with 0.7V volume AMpure beads.
- Nanodrop the purified DNA(optional) and stored it in -20C immediately.
Results[edit]
- 3uL of amplified product was validated using TBE-PAGE
- 3uL amplified libraries were validated using 6%TBE-PAGE with standard long staining method(250V, 20min).
- 3uL WGA amplicon + 3uL 0.5X TBE + 3uL 6X loading dye.
File:Sam080510- Nextera lib amp - phusion EZ.jpg File:Sam080610- Nextera-lib amplified.jpg
- The templates from these qPCR amplification is 10ng
- Start with 20ng but only 1/2 of 1-step product were amplified.
- The sample 10h-4 may contain more template than 90m-4 so it was amplified faster.
- The sample 10h-4 was prepared by dilution method to get estimated 20ng as starting amount. It is possible that the inaccuracy of quantification cause higher template used in 10h-4 sample.
- Using 10ng templates for qPCR is not stable in this method, even with 16cycle amplification the PCR amplicons still hard to be seen. However if I keep amplifying the background may start showing up.