cDNA Cloning Protocol

Digest the Cellecta cDNA or InDOXible vector with the corresponding restriction enzymes also used for preparation of cDNA fragments, and then verify complete digestion of the vector by agarose gel electrophoresis. If more than one restriction enzyme is used, we recommend that you skip the dephosphorylation step and perform only preparative gel purification of the digested vector. If you use a single restriction enzyme, dephosphorylation and gel purification of the digested vector is necessary in order to reduce the amount of background in the vector ligation step.

The optimal insert-to-vector molar ratio may be different for different inserts. Always try at least two different ratios (e.g., 10:1 and 30:1) for each experiment. Also make sure to include one negative control “no-insert” reaction, which contains only the digested vector.

Ligation of cDNA to Vector

1. Dilute the gel-purified digested vector to 10 ng/μl.

2. Set up 10 μl ligation reactions for each sample and control, as follows:
   

   Volume	Component
   1.0 µl	Digested cDNA or InDOXible Vector (10 ng/μl)
   x	cDNA insert (usually 15-50 ng)
   7.0 – x µl	Nuclease-free water
   1.0 µl	10X T4 DNA Ligase Buffer
   1.0 µl	T4 DNA ligase (40 U/μl)
   10.0 µl	Total volume

3. Incubate the ligation reaction at 16°C for 2-5 hrs, if it is sticky-end ligation. For blunt-end ligation, an overnight incubation time is recommended.

Transform competent cells with the ligation reaction

1. For each cDNA template, use the whole volume of ligation reaction for transformation.

2. Follow the manufacturer’s protocol for transforming the competent cells.

3. Plate an appropriate volume of cells on LB plates with 100 μg/ml ampicillin or carbenicillin and grow overnight at 37°C.

4. You should expect to get at least 10-fold more colonies in experimental samples in comparison with negative control (vector-only ligation reaction).

Identify clones with the target cDNA template

1. Prepare colony cultures.

2. Randomly pick up 10 well-separated colonies from each plate, and grow each clone in 100 μl of LB Broth with 100 μg/ml ampicillin or carbenicillin at 37°C for 2 hours with shaking.

3. Take 1 μl of each bacteria culture for PCR screening and continue to grow the culture for another 6 hours.

4. Store the bacterial culture at 4°C.

Screen for cDNA template inserts

1. Prepare a PCR master mix for each clone you would like to screen for the presence of a cDNA template insert as follows:
   

   1 rxn	10 rxn	Component
   0.5 µl	5 µl	Forward PCR Primer (10 μM)*
   0.5 µl	5 µl	Reverse PCR Primer (10 μM)*
   0.5 µl	5 µl	50X dNTP mix (10 mM each)
   2.5 µl	25 µl	10X PCR Reaction Buffer
   19.5 µl	195 µl	PCR-Grade Water
   0.5 µl	5 µl	Taq DNA Polymerase (5 U/μl)
   24.0 µl	240 µl	Total volume

*See the Product Certificate (PC) for your cDNA or InDOXible plasmid vector to find the required primer sequences.

2. Mix the master mix very well, and aliquot 24 μl into each well of a 96-well PCR plate or individual tubes.

3. Add 1 μl of each bacterial culture from B.1 into each well or tube from B.2.b. Mix.

4. Proceed with PCR using the following program:
   

   94°C, 4 minutes	1 cycle
   94°C, 30 seconds, 68°C, 1 minute	25 cycles
   68°C, 2 minutes	1 cycle

5. Take 5 μl PCR product and run it on a 3% agarose/EtBr gel in 1X TAE buffer.

6. Confirm identity of cDNA template inserts by sequence analysis of positive PCR products using the Forward PCR primer. Additional sequencing reactions with Reverse PCR Primer and/or cDNA-specific primers may need to be run if the insert is longer than your sequencing provider can read with good quality.

Purify Plasmid cDNA Construct

1. Take 15 μl of each positive bacteria culture, inoculate each clone in 10 ml of LB broth media with 100 μg/ml ampicillin or carbenicillin, and grow overnight at 37°C with shaking.

2. Purify cDNA construct plasmid DNA in Midi or Maxi scale using an Endotoxin-free plasmid purification kit.