Also note that, on the 335 resource page (link on the right) there are now a couple fo additional questions to practice assembling contigs, and an explanation on how to go about solving the RFLP question with the FCD.
Cheers
Pam
PS: remember that Friday (tomorrow) it's 'spring break'!
I can't seem to find the questions....
ReplyDelete-Jasmine
Try
ReplyDeletehttp://www.cfkeep.org/html/snapshot.php?id=40819551740605
Good luck!
Pam,
ReplyDeleteI have a question regarding the difference between making a cDNA library from RNA and using RT PCR.
In cDNA production,
You isolate your rna (how??) and you have RT which uses oligo(dt) as a primer and copies ONLY mRNA creating a hairpin loop. Your RNase H degrades the original strand and the DNA polymerase makes the second strand using the hairpin loop as the primer. Then you have Nuclease whihc degrades the hairpin loop. You now have double stranded cDNA and you throw this into vectors--> cDNA library. The end.......
With RT PCR
You have RNA and you use oligo(dT) to act as a primer for mRNA only. You add a 2nd primer with TAQ so that you only amplify the mRNA sequence you desire.....
So essentially the difference is that in one case..you're making a library...and in RT PCR you are looking for a specific fragment..how come no hairpin loops are formed in RT PCR?? Also, is it necessary to isolate the RNA initially or do we just filter all the junk out in the step when oligo (dT) is added as it only primers mRNA with polyA tails.....
You are trying to clone a 2.5kb EcoRI genomic fragment from your ET bacterium into the unique EcoRI site of a terrestrial 2kb plasmid that contains a gene conferring resistance to kanamycine. You have digested the plasmid with EcoRI, mixed it with your ET genomic DNA fragment, added the ligase and incubated the mixture as recommended by the (ligase) manufacturer. You then transform it into E.coli and plate the cells on complete medium kanamycine.
ReplyDelete32 colonies grow; you pick 12 at random, grow each one in liquid (complete) medium, and then proceed to plasmid extraction/purification. First, you digest a sample of each plasmid preparation with EcoRI and run each digestion reaction on a gel.
Here is a summary of what you get :
preps 1,2,4,5,6,9,10 have all the same pattern : 1 band at 2kb
preps 3,7,8,12 have all the same pattern :1 band at 2kb and 1 band at 2.5kb
prep 11 shows no bands at all
Explain these results! What can you say about the plasmids obtained in the different preps?
For the first set of preps I thought that the dna fragment did not successfully anneal with the plasmid and thus the cells contain an empty plasmid conferring resistance to Kan.
For the second set, is it possible that there were other EcoR1 cut sites on the plasmid where the fragment could anneal to......and kanR gene stays untouched so that the cell still has kan resistance???
Is it possible for a fragment to be transformed into a cell without a vector??? Perhaps it hopped in with the plasmid without actually inserting into the plasmid??
Is it possible that only a little bit of the KanR gene is disrupted...i.e. there are reduced levels of TXN ...but kanR resistance is still conferred..thus..these cells contain the vector with the inserted fragment.
for prep 11, is it possible that when you picked the colony......u killed it......???
Is it possible for a fragment to be transformed into a cell without a vector??? Perhaps it hopped in with the plasmid without actually inserting into the plasmid??
ReplyDelete*** i meant hopped into the cell without inserting into the plasmid
The RT-PCR and cDNA library questions...:
ReplyDeleteThere are several standard protocols to isolate RNA, and a number of tools and tricks to isolate only polyadelylated RNA.
What you described is correct; you can either run an RT reaction, make you cDNAs double stranded, collect them, and clone them into vectors (this way, you get a cDNA library); or you can run the RT and then, from the collection of cDNAs, amplify (by PCR) only your cDNA of interest, purify it and do whatever you want with it. The first method is to make a library, the second to detect your RNA (cDNA) of interest and possibly quantify it.
The hairpin loop formation is something that one can take advantage of (like in making the second strand of a cDNA WITHOUT knowing its sequence, and thus WITHOUT HAVING a second primer) or not (like when we have a hybrid RNA-cDNA at the end of an RT reaction and we want to use it in a PCR reaction, which implies that we DO KNOW parts of the sequence of our cDNA, and that we DO HAVE a specific primer for it).
The hairpin loop allows us to 'make double stranded' all the cDNAs in the mix.
I hopw this helps...
Cheers
Pam
About the cloning question up here...
ReplyDeleteFirst, be careful about the words, because it can cause misunderstandings: an insert does not anneal to a plasmid (rather, a single stranded probe, or a primer, anneal to the template, for example).
Fragments can't get into cells and stay in (so that we can purify them out and analyze them) without vectors: remember that linear fragments get degraded, and circular fragments with no origin of replication get 'diluted out'.
There can be as many restriction site as you like, so long as you don't contradict the information provided in the question. The question in this case is very specific about the presence of EcoRI sites. The question also does not say where this site is. I am not sure about your issues with the kanamycine resistance... what's the matter with that?
ALL the colonies analyzed contain the plasmid with an intact kan[r] gene, since they happily grow on a medium that has kanamycine in it, right?
For prep 11--if the colony had dies, you would see no growth in the liquid medium. We are not told that this happens (actually, we are told that we GROW each of the 12 colonies in liquid medium).
I hope these hints help...
Cheers
Pam
Hi Pam,
ReplyDeleteRegarding the radioactive bacteria question:
1) isolate the genomic dna of the new strain and perform partial restriction enzyme digestion.........choose an appropriate vector with (i.e. plasmid with ampR gene that stays intact and kanR gene that is disrupted when the plasmid is linearized and recombinated..).....linearize vector...mix partially digested dna fragments with linearized plasmid...ligate
2) transform the whole ligation mixture into kanS ampS radioactivityS E.coli........plate onto rich media + amp and then replica plate onto rich media + amp+ kan
The colonies which don't grow on the 2nd plate are the recombinant colonies.....
Grow recombinant colonies in Ampicillin and rich media and expose these colonies to radioactivity under otherwise sterile conditions.....and those that survive contain the radiactivity Resistance gene.
Then we isolate the plasmids and digest them to remove the fragments of DNA containing the gnee of interest..........
From this point...would we just analytically decipher the gene......i.e. look for Open reading frames...gene bashing to determine the entirety of the gene?
Also, is it possible to skip the entire step of replica plating...since the only colonies that will grow in radioactive conditions are those that contain the recombinant plasmid?
Pam,
ReplyDeleteDo we need to know about reading frames? My friend gave me a few old questions and they ask you determine the direction of transcription based on information about the reading frame? We havent really discussed this in class so is it something that won't be tested?
Hey Pam,
ReplyDeleteCould you post the answers to the loppin contig question on your questions page? I'm kind of unsure of my answer.
Hey everyone!
ReplyDeleteYou need to know what a reading frame is, and that's it. The answer to the contigs question is up, and the posted comment with the answer to the radioactive resistant bacteria is good (yes, you could skip the replica plating step!).
Cheers
Pam