Lecture 2 Flashcards
Why are Escherichia coli commonly grown in labs?
It’s a good way to produce plasmids and proteins through inserting it and then when it divides under the right conditions it will divide with the plasmid creating more of the plasmid or protenin that can later be used. It’s also a useful source of enzymes such as DNA polymerase, Klenow fragment and EcoRI. It can also be used as a host to phage P1, phage P4 and phage lambda.
How is phage P1 used in labs?
It can be used to transfer a selected mutation from one e-coli bacteria to another.
How is phage lambda used in labs?
It can be used as effective vectors to transfer recombinant DNA into another cell for cloning.
What does recombinant DNA mean?
It’s a cut and paste method used to insert genes of intrest.
What is a vector?
It’s plasmid based DNA that can be transferred to a new host cell.
How can phage P1 be used in labs?
It carries topoisomerase (specific type of enzyme) that can be used for cre recombinase which is a way to cut and paste insert genes of intrest. So it can be used for gene modification.
How are e-coli grown in labs?
They feed on peptone, yeast extract, NaCl, can grow in low oxygen levels which is perfect for lab work and grow best in 37 degrees.
What types of restriction endonucleases is there?
Those who leave blunt ends and those who leaves sticky ends. Type I cut at random far away from recognition sequences leaving sticky ends, type II is a homodimer (specfic type of large protein) and recognise the same DNA sequence, they can cut DNA both leaving blunt ends or sticky ends. Type IIS have asymmetric recognition sites leaving sticky ends.
What are some benefits with using plasmid DNA in the lab?
It’s coiled so can easily be seperated from the DNA, easy to amplify with bacteria cells in test tubes and can be purified with alkali denaturation lysis.
WHat do you need to keep in mind when growing plasmids in bacteria?
It’s need to be able to be recognised by the cell and be beneficial to the cell.
How does sanger sequencing work and why is it used?
It’s a way to determine the nucleotide sequence in the DNA. It works in several steps: 1.The double stranded DNA(dsDNA) is denatured into single strands DNA (ssDNA). 2.A primer that corresponds to the end of one of the sequences is added. 3. dNTPs which is a nitrigounus base binded to a sugar and trhee phostphate groups. There are four differenttypes for the different nucleotides and there are four different reactons happening at seperae each reaction with one of the dNTPs. 4.DNA synthesis initiates until a termination nucleotide is randomly incoperated. A termination nucleotide lacks the 3OH groups necessary to add another nucleotide to the chain. 5. The DNA fragments left is denatured into ssDNA and the denatured ssDNA fragments is run through gel electophoresis. Comparing the 4 different samples knowing that a dark band means that there was a base pair the sequence can be decided.
What is site directed mutagenesis and why is it used?
It’s a very site specific gene manipulation method that uses special primers in dsDNA to make insertions, deletions and substitutions. It’s a way to
How do we clone in lab?
It’s typically grown in E.-coi. There is a plasmid library, BAC library which is artificial chromosomes and colony hybridization which is when you grow and pick bacteria with the desired gene.
What is TA cloning and how is it used?
Instead of using resctrition enzymes it uses adenine and thymines ability to hybdridaze and in presence of ligase become ligated to each other. Using PCR product and vectors with overhangs enabled insertion of a gene into a plasmid.
What is a gateway cloning system and how is it used?
It’s a way to insert gene sequence into a plasmid without restriction enzymes or ligase.
What is a gibbson assembly and how is it used?
It is a cloning system that joins multiple DNA fragments in one single isothermal reaction. The steps are to first using PCR to create an overlap, adding an assembly mix that has axonuclease that chews back at the 5’ end creating an overhang and then the DNA polymerase will seal the gap.
