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===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 protocol'''). *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. No need for pipetting). *Shake the vials horizontally at 37C for 1 hour using vortexor in the incubator at the lowest speed. *Set up LB agar plates during the 1hr incubation. *After 1hr shacking mixing, plate '''50uL''' from each transformation vial on commercial LB agar plates (w/ Kanamycine, Teknova). **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. *'''NOTE: I plated 12 plates in total. Two plates per sample. Incubation started from 2:00PM 09-27-'10. Results (checked on 09-28-'10 12:00PM) Sample colonies count(white) NOTE ----------------------------------------- No.1-1 20 S1(0m) - No DNA Pol.I No.1-2 26 S1(0m) - No DNA Pol.I No.2-1 22 S1(0m) - W/ DNA Pol.I No.2-2 43 S1(0m) - W/ DNA Pol.I No.3-1 45 S1(30m) - No DNA Pol.I No.3-2 30 S1(30m) - No DNA Pol.I No.4-1 10 S1(30m) - W/ DNA Pol.I No.4-2 16 S1(30m) - W/ DNA Pol.I No.5-1 44 S1(60m) - No DNA Pol.I No.5-2 38 S1(60m) - No DNA Pol.I No.6-1 66 S1(60m) - W/ DNA Pol.I No.6-2 34 S1(60m) - W/ DNA Pol.I ----------------------------------------- *In general, the colonies number are fewer than I expected. It is possible that larger size insert is more difficult to clone. I will need to optimize the insert/vector ratio in the future. **The current ligation protocol works great for short inserts (200~400bp). For long insert (1kB) I may need to decrease amount to half for the reaction. '''I am curious if using only 8ng inserts can improve the results'''. *Except sample No.4, it looks like with S1 digestion treatment the successful cloning rate is a little bit increased. **The S1 digestion removes the single strand DNA, which leaves dsDNA for more blunting ligating.
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