Lab techniques

Question 1

how can you track what a cell differentiates into?

Answer 1

do cell lineage tracing using transgenic labelling. Can do this temporally using the cre-loxp system

Question 2

how can you view mRNA vs protein expression?

Answer 2

mRNA= mRNA
transgenic line (GFP)= protein

Question 3

how do you make a transgenic line in micr and zebrafish/ flies?

Answer 3

mice- homologus recombination: injected into blastula, chimera, gets in germ cells, offspring are het
zebrafish- inject the DNA into larvae and then will randomly integrate. Or pronuclear injection of oocyte then into mother.
flies- inject where the germ line is then breed (plasmid)

Question 4

how can you see if two proteins bind each other?

Answer 4

two hybrid system- fusion protein with two transcription factor sub domains (DNA binding domain and activating domain), that when together, will bind to express GFP on another GFP.

Question 5

what is the difference between transformation and transfection?

Answer 5

transformation is into non-eukaryotic cells, transfection is into eukaryotic cells

Question 6

what is ATAC seq and what is it used for?

Answer 6

it is a method of looking at areas of open chromatin structure within cells. A transpose cleaves and adds to adaptors to open regions, allowing amplification and sequencing of open regions.

Question 7

what is Chip (chromatin immunoprecipitation)?

Answer 7

The sample is exposed to a sample to UV or PFA which will induce cross linking of the transcription factor to DNA that it i sound to. Then you shear the cell and DNA using sonication. Then you pull down the transcription factor of interest by binding it to an antibody (immunoprecipitate). Then you unlink the DNA and sequence it. This will tell you where your TF binds

Question 8

How is a DNA microarray carried out?

Answer 8

You extract the RNA from your sample, then produce cDNA via RT-PCR. Then you label with a probe. Then you apply your sample cDNA to a DNA library of fixed probes. Then you can see the relative amounts of each.

Question 9

what is an alternative to DNA microarray and how does it work?

Answer 9

RNA seq- you extract the RNA and fragment and then convert to DNA that has adapters which allow it to be amplified and sequenced.

Question 10

what charge does DNA have?

Answer 10

negative

Question 11

what is the process of PCR?

Answer 11

separate, primers, extend, runnel

Question 12

what is a gene knock in? how can it be achieved?

Answer 12

can be achieved by homologous recombination,

Question 13

what is immunoprecipitation?

Answer 13

using an antibody to label a POI within solution and then precipitating it out

Question 14

what is a ligase?

Answer 14

an enzyme which binds DNA strands

Question 15

what is a western blot used for?

Answer 15

detecting levels of proteins within a sample

Question 16

what is a northern blot used for?

Answer 16

detecting levels of RNA levels in a sample

Question 17

if you want to amplify mRNA only, how do do this when designing the primers?

Answer 17

a primer that goes over an exon exon boundary- because introns are in DNA and won’t amplify if DNA if it has contaminated it

Question 18

how does sanger sequencing work?

Answer 18

have labelled nucleotides that stop sequencing. You then pull down strands of different lengths and look at the labelled nucleotide on the end and then can order them to get the sequence

Question 19

how does TaDa work?

Answer 19

enables tissue specific in vivo gene prolifing. You use damID but take advantage of at the fact that at a low frequency, eukaryotic ribosomes are able to reinitiate translation on bicistronic messages lacking an obvious IRES. So you put your dam-TFOI after a reporter gene and drive the expression of this using UAS/ Gal4 system. using the promoter of the TFOI to drive gal4- make two transgenic lines.

Question 20

what was the project in the Southall lab doing?

Answer 20

looking at the specification of neuronal subtypes in drosophila. They had already identified specific transcription factors for the three neuronal subtypes (cholinergic, glutamatergic and GABAergic) using Pol-2 and were then looking for these bound to.

Question 21

what is Pol-II?

Answer 21

it is DNA polymerase which transcribes genes when other transcription factors bind and activate it. So if you tag where this is binding in a cell type you will find what is being activated in this cell

Question 22

what is a drawback of TA-Da?

Answer 22

difficult and expensive to do in mice- two transgenic lines and cross- hard to check it is being expressed in the right place maybe- take longer