Molecular cloning exercise You used a TA cloning kit to clone your gene. An A wa

Question

Molecular cloning exercise You used a TA cloning kit to clone your gene. An A was introduced to the PCR product and T was matched with it in the vector pCR2.1 as shown. Your assignment is to subclone your gene from the pCR2.1 into the pAcGFP1-C1 vector. You have the MCS (multiple cloning sites) of each vector showing the different restriction digest sites. You also have the correct reading frame from the upstream GFP. You have to show what restriction enzyme sites you will use and when you clone into the GFP vector your gene will be in frame with the GFP and your fusion protein will be completely expressed. There are different ways of solving this problem. The more correct ways you describe the more points you will receive. Repeat the same exercise with the pEGFP-NI vector.

Explanation / Answer

To sub clone your protein of interest into pAcGFP1-C1, we compare the restriction sites provided on the vector as well as the lac z portion of pCR 2.1.

We look for restriction enzymes that are common to both our insert and MCS (upstream and downstream) and find that these combinations can be possible.

EcoR1 (upstream) and Xba1 (downstream)

EcoR1 (upstream) and Apa1 (downstream)

BamH1 (upstream) and Xba1 (downstream)

BamH1 (upstream) and Xba1 (downstream)

Sac1 (upstream) and Xba1 (downstream)

Sac1 (upstream) and Apa1 (downstream)

These restriction sites will ensure that our protien of interest will be coded into the new vector.

By using EcoR1 in the upstream, it will ensure that there will be less of junk sequence.

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