Question

Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids (now called a vector because it will carry your gene of interest) it is important to considering existing genes / DNA elements. If a site is in the middle of a gene, you could lose or destroy that gene. If there are multiple sites for an enzyme, when you paste them together, multiple possible outcomes can arise. This is undesirable, because it confounds verification of your desired plasmid. Sall EcoRI Vector Gene A Psti = plasmid that will carry your gene of interest Kpnl - 4,800 bp + BamHI Kpn Gene C Gene B BamHI Kpnl To demonstrate these principles, given the map above. Draw the results only as DNA fragments of the following digests below. Include the sites at the end of each fragment and all genes in their correct orientations. 1. EcoRI 2. BamHI If you were to ligate the BamHi digest, how many possible outcomes could you get? (just think about it, there are many if you consider it) For this reason, only unique sites that do NOT intersect a gene are used.

Answer 1

EcoR1 Ditgest Genec Genel Genea Sali Kpni Kpni Bambi Kpni BamH1 EcoR1 Pst1 BamH1 Digest Genec Gene B GeneA Genec BamH1 Kpn1 BamH1 EcoR Sali Kpn1 kpn1 BamH1 Pst1

If I was to ligate BamH1 Digest then 3 oucomes were possible.

There are 2 combinations for inserting the keratin protein one is through Pst1 - EcoR1 digest while the other is through Pst1- Sal1 Digest.

To make a GFP- Keratin Fusion Protein the cloning should be done through Pst1 - EcoR1 digest.

estu KERATIN Salt -ECORT -BAMHI Rank ken Orie KpnI

The GFP will be situated on the C-Terminal Side Of Keratin.