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Editors: Fatema Al Hashemi & Joanna Warren


Preparing the Cloning Vector
IMG_1005.JPG

What is a Cloning Vector?

“Cloning vector”: A DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment can be integrated without loss of the vector's capacity for self-replication.” (http://www.medterms.com/script/main/art.asp?articlekey=9801)

In this case, we are integrating a Rol-6 gene into an L4440 plasmid. The resulting cloning vector is then transformed into DH5α cells, which amplifies the desired plasmid in order to later be transformed into theHT115(DE) feeding strain so that the C.elegance worms behavior will alter.
molecular_l4440.jpghttp://www.biovisualtech.com/bvplasmid/L4440.jpg

The Rol-6 gene will be ligated between the SacII and HindIII restriction sites portrayed in the previous picture. Hence, after the collection of the L4440 vector and the Rol-6 gene the first step of creating the cloning vector would be the digestion of the L4440 vector with SacII and HindIII.



What is digestion?

Digestion is the process of using the chosen restriction enzymes, SacII and HindIII to cut open the plasmid so that later in the ligation process, the desired DNA fragment, which has one side cut with SacII and one side cut with HindIII, will be able to attach to the vector.

Procedure: (Because we had two separate PCR product batches, 2 separate ligations tubes were created).

PCR digest:
Tubes
10x Buffer
H20
SacII
HindIII
PCR product 1
PCR product 2
PCR 1 Digest
3ul
3ul
2ul
2ul
20ul
20ul
PCR 2 Digest
3ul
3ul
2ul
2ul
20ul
20ul

L4440 digest:
Tubes
L4440
SacII
HindIII
5x Restriction Buffer
ddH20
Lambda
L4440 digest
3ul
3ul
2ul
2ul
20ul
--
Control of HindIII
--
--
2ul
3ul
3ul
20ul
Control of SacII
--
2ul
--
3ul
3ul
20ul

Once all things were placed in the correct tubes, all the tubes were placed into a water bath at 37 degrees. After 20 minutes a gel was run to confirm that the PCR product and the L4440 vector were digested.

Lanes in gel:

Lane #1: 1KB ladder ( 5ul + 1 loading dye)
Lane #2: Uncut PCR (10 ul + 2 loading dye)
Lane #3: Cut PRC (10ul + 2 loading dye)
Lane #4: Uncut L4440 ( 10ul + 2 loading dye)
Lane #5: Cut L4440 (10ul + 2 loading dye)

Digestion of both was successful. Unfortunately, the gels that prove that digestion was successful have been misplaced.

The desired results for the digestion confirmation gel are:

Lane #1: 1KB ladder - muliple band
Lane #2: Uncut PCR - one band
Lane #3: Cut PRC - one lower band
Lane #4: Uncut L4440 - one band
Lane #5: Cut L4440 - one lower band

These results are similar to those we had on our gel, thus digestn was confirmed. Although we misplaced the gel, we do have a gel that confirms that the restriction enzymes, SacII and HindIII, worked.

Gel to Confirm HindIII and SacII Cut After 8 Hours of Digestion
gel_fin.jpg
The multiple bands in each lanes indicate that the restriction enzymes cut, which confirms that digestion was succesful.

Once digestion was confirmed, ligation was ready to be performed.



What is ligation?

Ligation is the process of attaching the desired DNA fragment into the vector. Because the two pieces were cut with the same enzymes, the SacII ends will attach together and the HindIII ends will attach together.

Procedure:

- Denature restriction enzymes at 65 degrees for 20 minutes then put everything in the correct tubes.


Digested L4440
Digested PCR Product #1
Digested PCR product #2
ddH20
T4 ligase
10x Ligase buffer
Ligated L4440 and PCR product #1
7ul
7ul
--
4ul
1ul
2ul
Ligated L4440 and PCR product #2
7ul
--
7ul
4ul
1ul
2ul

Once everything was in the tubes, the tubes were left at room temperate for 10 minutes. After the allotted time, a gel was run to confirm ligation.

Gel to Confirm Ligation

Gel_#2_fin.png
This gel did not confirm that ligation occurred because no bands appeared in the lanes besides the ladder lanes. We had not given the PCR produts and L4440 enough time to ligate together thus after 30 minutes, we ran run a second gel and the results confirmed ligation. Unfortuntely this gel has been misplaced.

Lane #2: 1KB ladder - multiple bands
Lane #3: Digested PCR product #1 - 2 bands
Lane #4: Digested PCR product #2 - 2 bands
Lane #5: Ligated tube #1 - 1 band, higher up on the gel the digested PCR bands
Lane #6: Ligated tube #2 - 1 band, higher up on the gel the digested PCR bands
Lane #7: 100bp ladder - multiple bands
Lane #8 Cut L4440 - 2 ban
ds

Because ligation was succesful, the vector had the DNA fragment (Rol-6) and the transfomation of the DH5a cells could now occur.