Please explain how to to determine the size of the bp indetail thanks alot The p
ID: 254943 • Letter: P
Question
Please explain how to to determine the size of the bp indetail thanks alot
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- overall size of pGEX-6P-2 is 4985 nucleotides.
Since the plasmid is circular so we will get to fragment when we digest plasmid with EcoRI and PstI.
the distance between EcoRI and PstI is 4132-1934 = 2198 Base pair and 2787 base pair.
We need to subtract the position of PstI from the position of EcoRI in the plasmid to get the size of the fragment.
2- since there is no site of PstI in the insert so we will get only two fragments upon digestion with EcoRI i.e. 55 bp and 235 base pair. fragments.
3- For PCR both the Primers i.e. Forward and reverse primers are needed. If we use only one primer then we will get the amplification of only one strand which will be the single-stranded DNA as the enzyme have only one template to make the copies. In this case, we will get a single-stranded DNA of 390 bases. The concentration the amplified single-stranded DNA will be very less
4- In this case, we will get the band of 390 base pair. Forward prime will bind at 762-780 base pair in the plasmid. The reverse primer will bind at 1151-1132. So when we subtract 1151-762 we will get 390 base pair