Molecular biology methods and experimental manipulation Flashcards
Restriction enzymes
Cut/cleave DNA often palindromic sections as they recognise specific sequences
Sticky ends
Parts left of when DNA is cleaved by an enzyme.
What sequence does enzyme Hipa1 find and where does it cut?
5-3’ GTT (CUT) AAC
What sequence does enzyme EcoR1 find and where does it cut?
5-3’ G. (cut). AATTC
What sequence does enzyme Hind111 find and where does it cut?
5-3’ A. (CUT). AGCTT
What sequence does enzyme Pst1 find and where does it cut?
5-3’ CTGCA. (CUT). G
What are Ligases?
Enzymes that stick compatible DNA back together.
If put in a test tube with sticky ends it will bind them back together with the use of ATP.
Globin Genes
Made in blood cells/red cells.
How is DNA cloned and amplified?
What is the most common?
In Microorganisms using Vectors (self-replicating genetic elements). Most common are those of E.coli Plasmids.
What are plasmids?
Double stranded circular DNA molecules that carry drug resistance genes, conferring resistance to certain antibiotics. Approx <20 kiloBase pairs (kb).
They contain an E.Coli origin of replication, when it copies DNA it also copies chromosomal sequence.
Also include gene of resistance (for example bla gene is ampicillin resistant).
2 outcomes of placing Ligase, DNA fragment and ATP in solution with plasmid and restriction enzyme?
Either plasmid joins back together to form original plasmid. Or accepts DNA fragment into plasmid.
What is a ligation mix?
When it is placed back into E.Coli what is it called?
ATP + ligase + DNA fragment + plasmid + restriction enzyme.
It is then put back into E.Coli.
Called transformation.
Agar Plates
Allow E.Coli to grow
Why would the E.coli cells fail to grow on ampicillin plates?
They did not accept the plasmid containing the ampicillin resistant gene.
Why would some plasmids not show their colour?
because the gene creating the blue would have been disrupted by accepting the DNA fragment.