TECHNIQUUES
I am expecting a lot of questions about techniques, mainly because most of you have never practically used any of them...
In the meantime, I am trying to put together some potentially useful info:
you can have a look here
(partly under construction, partly up and running)
In the meantime, I am trying to put together some potentially useful info:
you can have a look here
(partly under construction, partly up and running)
posted by pam39 at 8:00 PM
5 Comments:
Hi, Pam~ Hope you're enjoying Canada Day.
I still don't understand why you have to run a parallel gel when you do southern blot.
So you run southern and parallel gel at the same time. And you look for the gene of interest in southern and you cut plug of agarose in parallel gel. But why can't you cut plug of agarose in southern gel?
can we go over ORFs?
I was wondering why do we have to do complete digestion for plasmid DNA? Why can't we do partial digestion for plasmid DNA?
I know that for donor DNA we have to do partial digestion because we might cut our gene of interest if we do complete digestion.
Hi 335 people!
Shouldn't you be outside enjoying Canada Day, rather than studying for molecular genetics?
We can definitely go over ORFs. In a few words, they are stretches going from a start codon (AUG in the mRNA) to a STOP codon, and long enough to potentially encode a protein. Reminf me to talk more about this on Tuesday!
We need to run a parallel gel (parallel to the Southern) because the DNA that's on the gel used for the Southern will be blotted on the membrane (and it will stick there forever) so that we can do the hybridization with the probe. It is not possible to recover DNA that's stuck on a membrane.
If we just want to use the Southern for analytical purposes-which is usually the case-then we don't need a parallel gel. We only need it if we are trying to recover DNA from it.
Complete vs. partial digests:
the general rule is that whenever you are doing analytical digests, they have to be complete. Analytical digests are to see how many fragments we get, how large they are, etc...to analyze our clones/plasmids/constructs, to characterize genomic DNA using molecular markers, etc. if you do an incomplete digest when you are testing a construct, then you have meaningless/fuzzy results...let me know if this helps (or not)1
Cheers
Pam
Hi, Pam. Looking thru my notes, I have so many questions.
1. We can't not use RNA as probe because it's not stable as cDNA right?
2. How does the Northern Blot show splice variant??
3. mRNA is the only RNA with polyA tail right? (tRNA & rRNA don't have it?)
4. How could we amplify gene if we don't know the sequence?
We can use restriction enzyme to cut plasmid & amplify the plasmid DNA sequence (if we already know the plasmid sequence) Remember that drawing you drew on the board? You have gene in the middle and there are plasmid on either side of gene?
5. For RT-PCR, the cDNA has to express eukaryotic gene in bacteria right? We don't need to put enhancer, silencers to bacteria? So if we put cDNA into bacteria, it'll just work as prokaryotic gene regulation??
6. The difference between genomic & cDNA library is that: GENOMIC library is for making partial & complete library, cDNA library is produced at specific time of developmental stage. Am I correct?
5. We do partial digestion because we want to build genomic library because we don't know where the precise gene of interest is. We do complete digestion in order to do plasmid cutting right? But why is it analytical? To see how many bands we get on gel? To differentiate between homozygous vs. heterozygous individual?
Thank you.
I know it's a lot of questions. ^^
6. ORF question
-If we just find "STOP" codon, then it's close RF?
-If we just find "Start" codon,then it's open RF?
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