Step 4: End Repair and Cloning of the Sheared Genomic DNA

I prepared the following plates:

10 small plates (10 ml each) of LB media with carbenycilin

10 medium plates (20 ml each) of LB media with carbenycilin (200ul) and spectinomycin (200ul)

Concentrations of DNA extracted in step 3 were:

I tried to concentrate DNA a bit more in 25ul with a concentrator kit but the result was 3.7ng/ul so for the next steps I used 2 samples:

Sample 2: 7.0 ng/ul - 30 ul volume --> extracted from gel electrophoresis

Sample 3: direct result from the additional gDNA digestion I performed in step 3. I selected the Sau3 1:10 digestion

Mix the following components in a sterile microfuge tube (in all cases I used 19ul of purified DNA and 0ul of water since DNA quantity was lower than 2ug (we only did 2ug per digestion)

Reactions containing restriction enzyme digested DNA are incubated at room temperature for 15 minutes. Reactions with sheared/nebulized DNA or PCR products are incubated at room temperature for 30 minutes.

Immediately inactivate enzyme in the blunting reaction by heating at 70°C for 10 minutes.

Assemble ligation reactions using the chart below as a guide. Mix the first 4 components before adding 5 μl of the cloning mix consisting of 4 μl Cloning Mix 1 and 1 μl Cloning Mix 2, for a total of 10 μl per ligation reaction. This ensures the ligase is not allowed to recircularize the vector backbone before this insert is present. It is recommended that first-time users of this kit perform the positive control ligation reaction.

Incubate at room temperature (25°C) for 5–15 minutes. While 5 minutes is recomended, 15 minutes will increase transformation levels for inserts suspected as being difficult to clone.

Incubate on ice for 2 minutes.

Transform by electroporation into E. Coli ​E. coli electroporation

Recover cells for 1 hour

Plate 50 uL of the transformation onto an agar plate prepared with Carbenicillin and 200ul onto an agar plate with carbenicillin + spectinomycin. Incubate overnight at 37ºC.