Can explain how to do question 2 and 5. Not sure about question there as there i
ID: 257239 • Letter: C
Question
Can explain how to do question 2 and 5. Not sure about question there as there is an EcoRV in PCR mapping and not sure if need to look at PCR or PGEX or both. Thanks
The plasmids, primers, and DNA fragments you will be using in this assignment are . pGEX-6P-2 plasmid -this is a plasmid of the same family as the pGEX-2T used in the LEAPS practical:s. pGEX-6P-2-insert plasmid - this is the pGEX-6P-2 plasmid with a fragment inserted between the BamHI and Sal sites. This fragment is derived from a PCR product. . Forward Primer - this is a primer that has been previously designed for PCR that binds to the plasmid. Its location and orientation are shown on the plasmid map. . Reverse Primer - this is a primer that has been previously designed for PCR that binds to the plasmid. Its location and orientation are shown on the plasmid map. . GeneRuler1 kbp DNA Ladder-this is the DNA standard marker . The pGEX-6P-2 MCS and surrounding sequence.Explanation / Answer
1.EcoRV/Pst1 digest will give two fragments. Position of EcoRV is 4132 and that of Pst1 is 1934. EcoRV gives a blunt end digestion giving 3 bp on its 5' end and Pst1 gives a sticky end digestion with 1 bp at it 3' end The difference in terms of base pair is 2197
2.EcoRV is at position 55 and length of the fragment with blunt end digestion is 290 (size of insert)+ 220 bp (as EcoRV gives a blunt end digestion) =510 bp.The difference between Sal1 and Pst1 is 968 bp. Total size of the insert cut with these twwo enzymes are 1478 bp.
3.No fragment will be obtained without the forward primer only though sequencing reaction do use only single set of primers.
4.290 bp from the insert. Forward primer position (762), reverse primer position (1151 bp) and region of MCS removed (Sal1-BamH1 position= 965-945=20 bp). So the length of the sequence will be (1151-762-20)+290+1=661 bp