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Creation of Cloning Vector
Edited by Bryan Rehor

In this experiment, we needed to create a plasmid containing an exon of the dpy-10 gene: the gene that codes for normal worm growth. This recombinant plasmid creates dsRNA that is complimentary to dpy-10. We transformed this plasmid into the worms' feeding strain, HT155, so that when the worms ingested the bacteria, the also ingested the dsRNA, which triggered RNAi of this gene.

The plasmid L4440 was used as the basis of our recombinant cloning vector:


L4440_BR.jpg

In order to create the cloning vector, we needed to select multiple restriction sites that are 1) single cutters between the T7 promoters on the L4440 plasmid, 2) do not exist anywhere else within L4440, 3) not present in the insert, and 4) correspondent to restriction enzymes that retain activity when used together. The enzymes Sac II and Hind III meet these criteria as verified via the NEB cutter tool and double digest finder on the New England BioLabs website. NEB buffer #2 maximizes the enzyme activity. After we performed the restriction digest of the L4440, we combined it with the flanked dpy-10 gene, which was digested with the same two enzymes so that it had ends complimentary to the ends of our dpy-10 insert. In order to bind the two, we used T4 ligase in a standard ligation procedure.

Procedure:
We first grew a culture of cells that were known to contain the L4440 plasmid. To extract the plasmids, we performed a miniprep. Then, we did a restriction digest of the L4440 with Sac II and Hind III. In a 1.5 ml microcentrifuge tube, we combined the following reagents:

9 µl L4440
3 µl NEB Buffer #2
2 µl Hind III
2 µl Sac II
14 µl dd
H2O

After centrifuging, the tube was incubated for several hours on a 37°C. heat block. To confirm that the digest occured, we ran gel electrophoresis for 15 minutes at 300 volts. Observe that the L4440+ lane contains a band that is lower than the one in the L4440- lane, indicating that the plasmids must have been digested:
PCR.jpg


Because the dpy-10 PCR product was flanked with the two restriction sites, we needed to digest the PCR product with the same restriction enzymes in order to make the ends complementary with the restriction sites on the L4440. In a 1.5 ml microcentrifuge tube, we combined the following reagents:

15 µl dpy-10 PCR product
4.5 µl NEB Buffer #2
3 µl Hind III
3 µl Sac II
19.5 µl ddH2O

After centrifuging, the tube was incubated for several hours on a 37°C. heat block.

We denatured the restriction enzymes by heating each restriction digest tube to 65°C for 20 minutes. Then, we ligated the two restriction digests by combining the following reagents:

7 µl digested L4440
7 µl digested dpy-10 PCR product
4 µl ddH2O
1 µl T4 ligase
2 µl 10x T4 ligase buffer

After centrifuging, we incubated the tube at room temperature for 10 minutes. Then, we ran a gel electrophoresis of the ligated product, the dpy-10 PCR product, and the digested L4440 to confirm that the ligation occured. Observe the faint band in the ligation lane which does not exist in the L4440 digest lane, indicating that ligation has occurred.


ligation.jpg