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Help with mRNA purification and DNA polymerase!

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Help with mRNA purification and DNA polymerase!

Postby goose_keeper » Sun Oct 23, 2005 4:37 am

Goodday ,

Here are questions extracted from my exam paper, and I don't know
how to anwser perfectly. So anyone interested in the questions ,help
me please!

(1) Design a method to purify the mature mRNA in the eucaryotic cell.

(2) DNA polymerase has proofreading function, while RNA polymerase cannot proofread the wrong nucleotides. Explain the difference on the basis of biology.

LOOKING FORWARD YOUR REPLIES URGENTLY!
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Postby Morris » Sun Oct 23, 2005 11:58 am

I have a protocols about RNA extraction, but they're in Italian... If you're interested i can traslate in English.

I think that RNA polymerase hasn't proofreasding activity because, I think, RNA damaged or with wrong nucleotides is degradated by Rnases, but in DNA replication an error can bring dangerous mutations, that can be transferred to daughter cell. I think for this is important that DNA polymerse can correct immediately the wong nucelotide.
http://www.forumbiotech.forumfree.net(In italian)
http://www.forumfree.net/?c=37503 (international medicine forum)(In english)

http://u2forum.forumfree.net (in Italian)
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Re: Help with mRNA purification and DNA polymerase!

Postby sdekivit » Sun Oct 23, 2005 4:25 pm

goose_keeper wrote:Goodday ,

Here are questions extracted from my exam paper, and I don't know
how to anwser perfectly. So anyone interested in the questions ,help
me please!

(1) Design a method to purify the mature mRNA in the eucaryotic cell.

(2) DNA polymerase has proofreading function, while RNA polymerase cannot proofread the wrong nucleotides. Explain the difference on the basis of biology.

LOOKING FORWARD YOUR REPLIES URGENTLY!


the purification of mRNA can be done with affinity chromatography at high salt concentration, by using a poly dT group where mRNA can bindt to with it's polyA-tail.

The fact that RNA-polymerase doesn't have proofreading is that a mutation in RNA is degraded by the ubiquitin pathway and is just for 1 protein, whil a mutation in DNA is forever.
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Postby goose_keeper » Mon Oct 24, 2005 3:45 am

Thanks for your replies !

Morris said,
I have a protocols about RNA extraction, but they're in Italian... If you're interested i can traslate in English.

How to find your protocols since I can't speak Italian language?

To sedkevit
The Affinity chromatography is really an efective method for mRNA.
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Postby Morris » Mon Oct 24, 2005 1:03 pm

My protocols isn'ti n internet because comes foem an experiment, but I can pass it tp you by pm... When I have traslated it.
http://www.forumbiotech.forumfree.net(In italian)
http://www.forumfree.net/?c=37503 (international medicine forum)(In english)

http://u2forum.forumfree.net (in Italian)
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Postby canalon » Mon Oct 24, 2005 1:14 pm

goose_keeper wrote:How to find your protocols since I can't speak Italian language?


You will find all you need here:http://www.protocol-online.org/

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Postby Jelanen » Mon Oct 24, 2005 2:13 pm

Whoa..thats awesome...

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binding on the Affinity column

Postby VeeKAY » Thu Jul 10, 2008 10:39 am

I HAVE BEEN TRYING TO PURIFY MY GST-FUSION PROTEIN USING THE GLTATHIONE AGAROSE COLUMN AND AFTER RUNNING MY FRACTION SAMPLES ON SDS PAGE I ALWAYS SEE THREE BANDS INSTEAD OF ONE BAND ON MY ELUTED FRACTION. CAN YOU PLEASE HELP ME SO AS TO PURIFY MY FUSION PROTEIN AS A SINGLE BAND.
canalon wrote:
goose_keeper wrote:How to find your protocols since I can't speak Italian language?


You will find all you need here:http://www.protocol-online.org/

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Re: Help with mRNA purification and DNA polymerase!

Postby blcr11 » Thu Jul 10, 2008 12:02 pm

Invitrogen links to protocols-online. My country portal is the US so I get everything in English, but maybe if you enter through the Italian port to Invitrogen you get the protocols in Italian? Don't know.
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