Question

A1. The following is the DNA sequence of a hypothetical gene for the SMALL protein. It is called the SMALL gene. i atgggattac actgtcacga ccaaatagcc ttcattgtat 41 caaaaggato aatcgagtta tag Imagine you are doing a research project in a laboratory and your supervisor asks you to clone the SMALL gene into the PBR322 plasmid (shown below). You must use the Pstl and EcoRI sites for your cloning. HindIII EcoRI | EcoRV BamHI 4359 0 29 185 4000 375 Sall Psti 651 3607 amp tet 1000 PBR322 4361 bp 3000 ori 2000 2295 Ndel Your first step in the cloning is to cut PBR322 with Pstl and EcoRI (in the same tube) then electrophorese the cutting mixture on an agarose gel to examine the cutting efficiency. A picture of the gel electrophoresis equipment is shown on the next page.

Answer 1

A.Gel electrophoresis is a technique used to separate the DNA on their size and charge.Electrod A is negatively charged and Electrod B is positively charged.DNA fragments are negatively charged ,so they move towards to positively charged electrode(b),Because all the DNA fragments have the same amount of charge per mass ,small fragments move faster than the large ones.

B.Reverse primer:(with termination codon)

5'CTATAACTCGATTGATC3' GC=35% Tm-39.8C no self complementary ,no primer dimer

C.Polemrase Chain reaction(PCR) is a routine laboratory experiment which is used to amplify the particular DNA in test tube by using thernostable taqDNApolymerase,dNTPs,MgCl2,Primers,Buffers,DMSO and template.The PCR generally runs in various steps such as Denaturation,Elongation,Extenstion and these are occurs at high temperatures.So Taq DNA polymerase is thermostable enzyme from thermus aquaticus and its withstand at high temperatures   

D.The gene encodes 20 aminoacids they are MGLHCHDQIAFIVSKGSIEL .Because they are 60 basepairs and one stop codon so one aminoa acid is encoded by the three basepairs.