Entry Vector Cloning Protocol

The open reading frame (ORF) of the clone must be PCR amplified in order to clone the ORF into an Entry Vector. Please view the Primer Design and PCR Amplification of ORFs Protocol before moving forward.

Step 1, Electrophoresis:

1. Confirm the size of the amplification product by agarose gel electrophoresis and purify the remainder of the reaction using a purification column or similar method.

Step 2, Digestion:

1. Elute the DNA from the purification column in 26 ?l of 10 mM Tris buffer. Set up a digestion reaction as described below, substituting other restriction enzymes as appropriate.
   Component	Volume
   10X restriction buffer	3 ?l
   Sgf I (10U/?l)	0.6 ?l
   Mlu I (10U/?l)	0.6 ?l
   Purified PCR product	26 ?l
   Total volume	~30 ?l

   Mix well, and incubate at 37?C for 3 hrs.

2. Purify the digestion reaction using a purification column and elute in 18 ?l of 10 mM Tris buffer.
3. Quantitate the DNA by UV at A260.
4. Digest the pCMV6-Entry vector with the restriction enzymes corresponding to the sequences added to the ORF. The pCMV6-Entry Vector is available from OriGene as 10 ?g lyophilized DNA (Cat# PS100001). Resuspend the lyophilized DNA in 100 ?l dH2O, and incubate for at least 30 min before use. Set up a digestion reaction as described below, substituting other restriction enzymes as appropriate.
   Component	Volume
   10X restriction buffer	3 ?l
   Sgf I (10U/?l)	0.8 ?l
   Mlu I (10U/?l)	0.8 ?l
   Nuclease-free water	15.4 ?l
   Vector DNA	10 ?l
   Total volume	30 ?l

   Incubate at 37?C for 3 hrs, then add 1 ?l antarctic phosphatase (units used according to the manufacturer?s protocol), and continue the incubation at 37?C for another 30 min. Dephosphorylation of the digested vector is essential to eliminate self-ligation.

5. Purify the desired vector fragment by running the digestion reaction on an agarose gel, and isolating the appropriate band using a gel purification column. Elute the digested plasmid vector in 40 ?l of 10 mM Tris buffer.

Step 3, Ligation:

1. Set up a ligation reaction with the purified vector and insert fragments:
   Component	Volume
   10X ligase buffer	1?l
   Nuclease-free water	3.5?l
   T4 DNA Ligase	0.5?l
   Vector Fragment	2?l (approx. 10ng)*
   PCR product	3?l (approx. 30ng)*
   Total volume	10 ?l

   Incubate the ligation reaction at room temperature for 30-60 minutes. * Alternate ratios may need to be tested to obtain optional ligation efficiency.

Step 4, Transformation:

1. Transform 1 ?l of the ligation mixture using 20 ?l high efficiency competent E. coli cells (ideally 1x108 CFU/ug). Following transformation, resuspend cells in 200 uL LB.
2. Plate the entire transformation reaction on a standard LB-agar plate containing 25 ?g/ml kanamycin. Incubate at 37?C overnight.
3. Pick at least 4-8 independent colonies to do miniprep from each ligation. Confirm the insert by restriction digestion and/or vector primer sequencing.