The+Creation+of+the+Cloning+Vector+Using+L4440

Home Page =// Creation of the Cloning Vector Using L4440 //= // What is a cloning vector? A vector serves to transport fragments of DNA into a host cell. Specifically, cloning vectors are used in order to make multiple copies of an inserted piece of DNA. Upon digesting the cloning vector and the DNA fragment to be incorporated with the same restriction enzymes, one can continue to ligate, or join, the vector and the foreign DNA. In order to ensure successful insertion of DNA into cloning vectors, most of the carriers have their restriction enzyme sites removed. To replace these distinct restriction sites, a Multiple Cloning Site (MCS) is engineered into cloning vectors. MCSs are comprised of a string of restriction sites.

Why use L4440? // In our RNAi experiment, use of the L4440 cloning vector (pictured above) will hopefully result in successful recombinaton of the plasmid and our gene of interest, Blister-1. Conveniently located to control the expression of our target DNA, the two T7 promoter sequences, which originate from the bacteriophage T7, flank the MCS. By choosing two enzymes that are compatible in a double digest and that both have restriction sites in the MCS, we can open up the L4440 plasmid, thereby preparing it for ligation with the PCR product, which should contain our gene of interest. For our purposes, we have chosen restriction enzymes HindIII and SacII to digest both our cloning vector and our PCR product. Choosing two different enzymes to cut within the T7 promoter was key. Using only one restriction enzyme to digest the L4440 vector would have created a higher probability of the sticky ends annealing to one another, and thus, the plasmid would close before it could receive the gene of interest.

Effective ligation of the L4440 cloning vector and the Blister-1 gene will allow for the subsequent transformation of DH5alpha cells and will bring us one step closer to gene silencing in our //C. elegans//! // Procedure and Results //  1. 2 cultures are needed in order to make four separate mini-preps:  2. Place the cultures in a 37 degree Celsius shaking water bath for 24 hours.
 * LB tubes
 * AMP (1,000 x) 1 uL/mL
 * ~500 uL L4440 (in DH5alpha) per culture

3. Conduct 4 Zippy mini-preps to separate the L4440 plasmid from the //E. coli // DH5alpha strain host cells.

4. Digest the L4440 vector using the following protocols. Place in a 37 degree Celsius water bath for several hours.
 * 30 ul L4440 plasmid
 * 2.5 ul HindIII
 * 2.5 ul SacII
 * 5 ul NEB Buffer #2 (Click on the link to be directed to the NEB website double digest finder for HindIII and SacII).
 * <span style="font-family: Arial,sans-serif;">10 ul ddH2O

5. Run a gel to confirm that the four mini-preps and the digestion of L4440 were successful. <span style="font-family: Arial,sans-serif;"> 6. Digest the PCR product using the following protocol. Place the digestion tube in a 37 degree Celsius water bath for several hours.
 * 20 ul Bli-1 PCR product
 * 2 ul HindIII
 * 3 ul SacII
 * 3 ul NEB Buffer #2
 * 2 ul ddH20

7. Run<span style="font-family: Arial,Helvetica,sans-serif;"> a gel to verify that the digestion of PCR was successful and that HindIII and SacII are functioning properly (i.e. cutting). <span style="font-family: Arial,Helvetica,sans-serif; font-size: 10pt;">

8. If both the L4440 plasmid and the PCR product have been digested properly at this point, denature the restriction enzymes by placing the two tubes in a 65 degree heat block for 20 minutes.

9. Combine the two separate digests in a Ligation tube using the following protocol.
 * 16 ul digested L4440 plasmid
 * 16 ul digested PCR product
 * 2 ul ddH2O
 * 2 ul T4 ligase
 * 4 ul 10x T4 ligase buffer

10. Ligate at room temperature for 20 minutes.

11. Run a gel to confirm successful ligation.

//Sources//

http://en.wikipedia.org/wiki/Cloning_vector http://en.wikipedia.org/wiki/Multiple_cloning_site