GRK2 Subcloning Project

Current Status

Redoing step 1

Protocol

Locate plasmids in SnapGene Files

1. Create a plasmid map of GRK2 in the pGHE backbone using the TAAR1_pGHE plasmid and the coding sequence for GRK2 from DNASU. The coding sequence for GRK2 will simply replace the TAAR1 protein coding sequence.

2. Design primers from this plasmid map, four total. A GRK2 forward HiFi primer ~25nt in length and a GRK2 reverse HiFi primer of similar length, this will result in amplification of the GRK2 coding sequence for pGHE. For amplification of the pGHE backbone for GRK2, a GRK2_pGHE reverse primer ~25nt in length and a GRK2_pGHE forward primer of similar length. These primers should be arranged for a ~10-15bp overlap and will overlap in pairs like so: GRK2 forward/GRK2_pGHE reverse, and GRK2 reverse/GRK2_pGHE forward. Protocol to help with this found here at the top of the page.

3. Order the protein coding sequence in a plasmid of choice from DNASU and primers from New England BioLabs.

4. Send the primers and plasmids, once received from DNASU to GeneWiz for sequencing.

5. Once the sequences are confirmed, PCR can be run.

6. Two PCR reactions will be ran, each using one set of primers from step 2 (GRK2_pGHE forward and reverse/ GRK2 forward and reverse). These reaction mixtures can be made and run at the same time. The first reaction mixture will amplify the pGHE backbone and will contain the source of pGHE backbone (TAAR1_pGHE or B2AR_pGHE) and the primers of this backbone that are specific for GRK2, designed in step 2 (GRK2_pGHE forward and reverse). The second reaction mixture will amplify the GRK2 coding sequence and will contain the GRK2 DNA template (purchased from DNASU, step 3), and the primers for this coding sequence in pGHE designed in step 2. PCR protocol found here.

7. PCR products are then analyzed through gel electrophoresis or by the GeneWiz company. Protocol for running and analyzing a gel can be found here.

8. Phosphorylation and ligation of two PCR products into circular DNA. Protocol found here.

9. Circular DNA product then multiplied through transformation by JM109 E. Coli bacterial cells. Protocol found here.

10. If colonies form from bacterial transformation, single colony inoculation is ran. This must be coordinated for a mini-. midi-, or maxi-prep and will define the total volume of the reaction mixture to be inoculated. The goal of this process is to now grow the bacteria in a liquid media rather than a solid agar plate media to facilitate the extraction of DNA after. See protocol for single colony inoculation for details.

11. Inoculation products will then have DNA extracted and isolated through a phenol-chloroform extraction and ethanol precipitation of the DNA. The goal of this process is to obtain a DNA "pellet" in the bottom of the reaction tube following centrifugation. This process will only vary by volume for a mini-, midi-, or maxi-prep. See DNA extraction and precipitation protocol for more details.

12. DNA is then linearized from its previous circular form to facilitate transcription into RNA. Linearization protocol found here.

13. Linearized DNA is transcribed in vitro to RNA. Protocol will be found here. This RNA product can then be stored in the -80 degree freezer downstairs until it is ready for injection into an oocyte.