Sam:LabNotes/Microbiome-new/2010-7-10

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Blunt cloning sequencing of barcoded-Solexa libraries (made by library-free protocol with Dinh)[edit]

Background[edit]

  • Twelve Solexa libraries were made using barcoded-primer with Solexa sequencing tails (By Dinh). The templates were pad-lock probe captured inserts.
  • Two libraries were sequenced using cloning sequenincg.
  • Since the PCR products were generated using Phusion enzyme, there is no need for doing blunting on these PCR products. The PCR product could be used directly for blunting ligation.

Blunt-vector ligation[edit]

  • Invitrogen Zero-blunt PCR Cloning kit (Invitrogen)
  • Set up ligation reaction in PCR tubes on cold block:
Sam's protocol (Invitrogen manual protocol)

                                        1rxn     
   PCR product of seq. lib (~15ng/uL)   3.0 - Since each template is about 4ng/uL       
   10X Ligation buffer                  1.0     
   pCR-Blunt (25ng/uL)                  1.0     
   RT-PCR grade H2O                     4.0
   (Red cap)T4 DNA ligase (4U/uL)       1.0     
   -----------------------------------------
                                       10.0  
  • Incubate at 16C for 1hr using PCR thermocycler. Perform the transfection using the ligation product as soon as possible.

Heat-shock transfection[edit]

  • Equilibrate water bath to 42C.
  • Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
  • Thaw the TOP10 E.coli competent cell vial on ice.
  • Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocl).
  • Set up conpetent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins.
  • Adjust the timer: set up 30sec and 2min.
  • Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
  • Add 250ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall).
  • Shake the vials horizontally at 37C for 1 hour using vortexor in the incubator at the lowest speed.
  • During the 1hr incubation.
  • After 1hr shacking mixing, plate 50uL from each transformation vial on agar plates (w/ Kanamycine).
    • There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design.
  • Incubate plates at 37C for about 20hr.

NEXT DAY

Results[edit]

  • D1: Library-1 sample
  • D2: Library-2 sample
  • Except D1-3 and D2-3, the other reactions were plated on pre-made agar plate (w/ Kanamycine)
                     D1-1       D1-2      D1-3(Sam agar plate)      D2-1       D2-2       D2-3(Sam agar plate)
--------------------------------------------------------------------------------------------------------------
White colonies       20         0         46                        75         6          135

Genewiz sequencing sample preparation[edit]

  • Four colonies from each libires were picked and innoculated into 10uL H2O to make new templates for clonyl PCR reactions.
    • These templates are denatured at 94C for 10min and frozen in -20C before use.
    • The clonyl PCR reactions were performed successfully. The PCR products were purified and sent for Sanger sequencing (by Dinh).
  • Two agar plates (one from each library) were sent to Genewiz using "Sequencing bacteria colonies" service.
    • Two colonies from each agar plates will randomly picked up for sequencing.

Results[edit]

  • Only six out of eight reaction (Clonyl PCR products) were successfully sequenced.
    • The other were failed by no priming with unknown reason.
  • The sequences were trimmed and only the last 100nt sequences were kept.
  • The trimmed sequences were multi-aligned to find out primer and barcode region:
File:Sam071310-Library validation-new.jpg

  • The results 100% match the reverse complementary sequences of the designed barcodes
    • Barcode index 1: 5'---CGTGAT---3'
    • Barcode index 2: 5'---ACATCG---3'
  • Actually the clonyl PCR is a very good method to validate the barcoded libraries.
  • This test only includes the first two barcorded index-primers, more index-primers (no.3 ~no.12) will be tested on more other captured products (made by Dinh).