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='''Blunt cloning sequencing of barcoded-Solexa libraries (made by library-free protocol with Dinh)'''= ==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 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== *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|1100px]] *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).
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