Sam:LabNotes/Microbiome-new/2010-7-10: Difference between revisions

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>Sam Chiang
(Created page with '='''Blunt cloning sequencing of Barcoded-Sol library (Library-free protocl test)'''= ==Background== *Twelve Solexa libraries were made using barcoded-primer with Solexa sequenci…')
 
>Sam Chiang
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*Incubate at 16C for 1hr using PCR thermocycler. Perform the transfection using the ligation product as soon as possible.
*Incubate at 16C for 1hr using PCR thermocycler. Perform the transfection using the ligation product as soon as possible.


===Heat-shock transfection===
===Heat-shock transfection===
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**There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design.
**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.
*Incubate plates at 37C for about 20hr.
'''NEXT DAY'''


==Results==
==Results==

Revision as of 08:24, 13 July 2010

Blunt cloning sequencing of Barcoded-Sol library (Library-free protocl test)

Background

  • 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

  • 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

  • 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

  • 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

  • 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 PCR reaction were performed successfully. The PCR products were sent for Sanger sequencing.
  • 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 equencing.