Conventional Cloning

Conventional Cloning

Gene insert preparation

(Note:​​ conventional cloning can only insert one gene at a time)

• Primer design: Design the gene-specific forward and reverse​​ primers with the desired two restriction digestion site plus extra nucleotides for different restriction sites (see the “extra nucleotides for restriction sites” pdf file) to facilitate enzyme binding.

• PCR amplify the gene of interest

• Optional: if the template used is a plasmid with the same antibiotic marker as the cut vector, proceed to DpnI treatment to digest it.

• Purifying the PCR product using either the PCR purification kit or gel purification kit

Restriction digestion

• For the purified PCR product:

Recipe:

PCR product  : 16 ul

10 X NEB buffer : 2 ul (buffer may differ with different NEB enzymes)

Restriction enzyme 1 : 1 ul

Restriction enzyme 2 : 1 ul

Total volume  : 20 ul

Maintain 37˚C overnight, purify the digested PCR product and measure DNA​​ concentration using Nano drop.

• For plasmid backbone treatment:

Recipe:

Plasmid  : up to 1 ug

10 X NEB buffer : 5 ul (buffer may differ with different NEB enzymes)

Restriction enzyme 1 : 1 ul

Restriction enzyme 2 : 1 ul

ddH2O   : to make up for a 50 ul​​ reaction

Maintain 37 ˚C overnight, add 10x Antarctic phosphatase buffer: 5.5 ul, and 1 ul Antarctic phosphatase, into the digested plasmid above, incubate at 37 ˚C for 15 minutes, then incubate the reaction at 65 ˚C for 10 min to inactivate the phosphatase. Purify the cut vector using the PCR purification kit and measure DNA concentration using Nano drop.

Ligation:​​ 

Recipe:

Cut vector: 10 ng

Cut insert: to make up 10 ul reaction (negative control: use H2O to make up 10 ul reaction)

10 X ligation buffer: 1​​ ul

T4 DNA ligase: 1ul

Maintain at room temperature for 3 hours, Transform 1 ul ligation mixture to DH5a chemically competent cells. Grow on LB plus antibiotic at 37 ˚C overnight. There should be more colonies on the plate with the insert than the​​ negative control without.