Gene Cloning Tools Flashcards
Describe how the vector DNA had to be prepared for ligation?
Purify with alkaline lysis, digest with restriction enzymes and treat with alkaline phosphatase to remove the 5’ phosphates.
Describe how the target DNA had to be prepared for ligation?
Purify, PCR amplify with designed primers and digest with restriction enzymes.
What occurs after ligation of the vector and target DNA?
The ligation mix is transformed into competent E.coli, plate into an agar plate with an antibiotic, screen colonies to identify those with recombinant plasmid- colony PCR or plasmid isolation and restriction digest.
How many DNA molecules dies each cell take up during transformation?
1 per cell
How is cDNA made?
From mRNA using reverse transcriptase and a primer.
What primers are used for cDNA synthesis?
For first strand synthesis usually an oligo dT and for second strand synthesis usually a self-priming or specific primer plus DNA polymerase
What is PCR?
Polymerase chain reaction: a method of DNA amplification
How many cycles of thermal cycling does PCR require?
25-40
Describe a PCR thermal cycle?
90 oC initial denaturation, 55 oC anneal primers, 72 oC DNA synthesis (extension).
What enzymes are required for PCR?
Thermostable DNA polymerases
Give an example of some thermostable DNA polymerases?
Taq: 5’ -3’ exonuclease and DNA synthesis
Kod, Pfu: 5’-3’ DNA synthesis and 3’-5’ proof-reading exonuclease.
During PCR how does the DNA get amplified in each step?
Exponentially
What has to be considered when designing a primer?
Length, GC content, melting temperature, complimentarity, polypyrimidine/polypurine sequences.
What length should a suitable primer be?
~20 nucleotides long
What GC content should a suitable primer have?
~50%
What melting temperature should a suitable primer have?
Similar for both, > 55 oC
Why is it important to assess complimentarity of primers?
As you do not want them to be complementary to one and other.
Why is it important to assess polypyrimidine and polypurine content of primers?
As you do not want stretched of either.
What can you do to the 5’ end of primers?
Add sequences.
What are some common cloning vectors?
Plasmids, viruses/bacteriophage, cosmids and phagemids
What is a cosmid a combination of?
Plasmid and bacteriophage lambda
What is a phagemid a combination of?
Plasmid and bacteriophage M13
What do vectors used for cloning usually have?
Multiple cloning sites, promoter, origin of replication, antibiotic resistance, a method (eg. Tag) for purification.
What are restriction sites?
6 nucleotide stretches of DNA which are recognised by restriction enzymes and ensure cleavage occurs on both the vector and target DNA in order for ligation.
How is agarose gel electrophoresis used to analyse DNA fragments?
DNA and RNA have a net negative charge due to phosphate groups so they are attracted towards the anode. Larger molecules move slower through the matrix and smaller molecules faster.
What sort of ends can restriction enzymes leave?
Blunt or sticky (overhang)
Before transformation what are E.coli cells treated with and why?
CaCl2 or RbCl2 to disrupt their cell walls
How are E.coli cells encouraged encouraged to take up the DNA?
By heat shock
Why when plated on agar with antibiotics do only clones that contain the vector grow?
As they encode antibiotic resistance.
What are some key molecular biology tools?
Vectors Agarose gels Restriction enzymes Modifying enzymes Ligases Polymerases Synthetic DNA Polymerase chain reaction (PCR)
