You have identified a new gene of interest which is described as follows 5\' GAA
ID: 282031 • Letter: Y
Question
You have identified a new gene of interest which is described as follows 5' GAATTCTGCCTTAAAATTTGTTTGTGGGATG.... 4kb of DNA sequence... TAAGTTTCTGTAAATGTGTAAAAGTCGAC-3. You wish to insert the gene into the blue script vector. What restriction enzymes would you use? Describe the process of generating recombinant DNA. What features are required in the plasmid to select for positive recombinants? The purple gene is Lacz. How does this aid the identification of positive recombinants? You perform two digests. One with EcoRI alone and one with EcoRI and BamHI. Describe the results you would get on a aggarose gel after the reaction goes to completion. aErprom(1000%! F1 intergenic region Nael (132) Scal (2564 M131 (1000%) APr T7 promoter Sac ll (662) ?No1 (668) EcoRI (734) pBluescriptR MCS Sall (740) Xhol (745) 2998 bp BamHI (754) Apal (792) Acc651 (794) Kpni (798) uromotor 194.1 % AspEI (2084) pUC oriExplanation / Answer
The vector contains MCS (multiple cloning site) containing restriction sites for variety of enzymes. Any of these restriction enzymes can be used keeping in mind that this site should not be present in the gene to be cloned otherwise the target gene will also get digested.
Process of generating recombinant DNA
1.introductionof target DNA into a suitable cloning vector which is here pBLUESCRIPT.
Extraction of target DNA from source which is in this case the unknown gene given ,This gene can be PCR amplified and then purified PCR product can be used.
The vector as well as DNA is restriction digested and then the target DNA can be ligated onto the vector by the action od DNA ligase.
The resulting ligated product is further confirmed for the presence of target gene/DNA by using gene specific primers.
This ligated product is the transformed into competent bacterial strain.
Plating of bacterial cells onto selection plate containing antibiotic resistance genes
Selection of recombinants bacterial colonies.
Vector should have following properties for selection of recombinants-
1.Generally the plasmids possess antibiotic resistance gene which confers resistance to particular antibiotic. Thus the cells which gets transformed by the plasmid will become resistant and therfore can be selected onto the plates containing antibiotic whereas the other which do not carry plasmid will be sensitive and will not survive in presence of antibiotic.
2. Lac Z gene codes for enzyme known as beta- galactosidase, This is involved in breakdown of X gal (substrate LACTOSE ANALOG), which is acted upon by beta -galactosidase to produce blue colored colonies.
if the DNA/GENE is inserted into lac Z, the gene will get inactivated and therfore functional beta galactosidase will not be produced. Thus the X gal will not be hydrolysed and the colonies remain white.
Thus recombinants caan be selected from non recombinants based on the blue-white color selection.
Results
Digestion of recombinant DNA with EcoRI alone produce 2 cuts one in the vector DNA and other in the target DNA . 2 bands will be produced one of vector DNA and other the insert DNA.
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.