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cDNA synthesis


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#1 cheerioet

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Posted 27 May 2009 - 06:56 AM

In need of help...Thanks...

I am currently dealing with extracted RNA that needed to be further used to 1st Strand cDNA synthesis.

I am using Qiagen MinElute Kit to extract the RNA and I did not treat the eluted RNA with RNase Inhibitor.

The question I want to ask is that, after I eluted the RNA, how do I treat the extracted RNA with RNase Inhibitor?

And, the 1st strand cDNA synthesis that I am doing was fine for the control positive provided by the kit, but not my sample RNA.

I would also want to continue a PCR amplification for the synthesized 1st strand cDNA, according to the manufacturer's protocol for the control positive, they provided a forward primer and reverse primer; but as for my case, I do not have specific primer, because I wanted to get the possibility of the whole cDNA amplified-I used random primer instead.

And do anyone have the experience in doing the same thing?

I need the amplified cDNA to do RE digestion and to get them cloned.

Would appreciate anyone's help over here, and thanks in advance.

#2 bob1

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Posted 27 May 2009 - 04:41 PM

If you are looking to clone a specific gene, I recommend using a RACE (rapid amplification of cDNA ends) kit. I would also recommend using an oligo dT based prep of your cDNA unless you know your gene does not have a poly A tail.

If your technique is OK, you shouldn't need RNase inhibitor treatments. The kit protocol suggests that if you do need them , to add them before the extraction at the optional B-me step..

#3 cheerioet

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Posted 27 May 2009 - 06:57 PM

If you are looking to clone a specific gene, I recommend using a RACE (rapid amplification of cDNA ends) kit. I would also recommend using an oligo dT based prep of your cDNA unless you know your gene does not have a poly A tail.

If your technique is OK, you shouldn't need RNase inhibitor treatments. The kit protocol suggests that if you do need them , to add them before the extraction at the optional B-me step..



Hi Bob,

I am actually did not have any specific gene as well, because we wanted to try to fish out every possible cDNA synthesis. Will oligo dT is better? Well, after the 1st strand of cDNA synthesis using the oligo dT, can I use random primer or what type of primer will be suitable for the PCR amplification? I do not have specific primer in this case, because I need to have the possibility of getting any cDNA amplification to use it to do the RE digestion for cloning.

As for the RNase inhibitor treatment, after I eluted the RNA using the spin column from the kit, will I still need to phenol chloroform precipitate it? Or I can directly add in RNase Inhibitor unit in the proportion recommended by the manufacturer and incubate it, then the incubated RNA with RNase Inhibitor can be directly used for cDNA synthesis?

Thanks in advance for the suggestion and answer.


#4 Rsm

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Posted 27 May 2009 - 10:06 PM

We used to do some similar stuff from single cells, technology is similar to PMID 16877544 (search this number in PubMed). You would use an oligo dT primer first, then end-label your cDNA with dA and terminal transferase and use oligo dT primer again. You can use RE sites fused to your oligo dT.
However, from my experience, it is very difficult to get full-length cDNA with this approach. We used to clone the PCR products and sequence the clones. If you've got something interesting, make specific primers. Secondly, most clones we had were fusions of different mRNAs, due to sticky ends. You'll need a good program to sort them out... (Phred?)
No, you don't need to Phenol precipitate your mRNA after elution, just adding RNase inhibitor is sufficient. You can use that directly for cDNA synthesis.

Cheers,
Minna
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#5 cheerioet

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Posted 28 May 2009 - 06:15 PM

We used to do some similar stuff from single cells, technology is similar to PMID 16877544 (search this number in PubMed). You would use an oligo dT primer first, then end-label your cDNA with dA and terminal transferase and use oligo dT primer again. You can use RE sites fused to your oligo dT.
However, from my experience, it is very difficult to get full-length cDNA with this approach. We used to clone the PCR products and sequence the clones. If you've got something interesting, make specific primers. Secondly, most clones we had were fusions of different mRNAs, due to sticky ends. You'll need a good program to sort them out... (Phred?)
No, you don't need to Phenol precipitate your mRNA after elution, just adding RNase inhibitor is sufficient. You can use that directly for cDNA synthesis.

Cheers,
Minna



Erm...Thanks Minna for the advise.

I am now still having problem in synthesizing the 1st strand cDNA. Trying to get fresh phage to extract for RNA and redo the 1st strand cDNA again. The problem that I am facing now also is the lack of reagents left in the previously left over kit.

"end-label your cDNA with dA and terminal transferase and use oligo dT primer again"

Can I use oligo dT to synthesis 1st strand cDNA and then use the Random primer to amplify it?

Thanks.


#6 Rsm

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Posted 29 May 2009 - 09:34 PM

Oh, my answer was not on topic, sorry...
Wait, you said you want to "get fresh phage to extract for RNA", does this mean you're working with bacteria phage RNA? Do you know if that is polyadenylated? AFAIK, bacteria don't polyadenylate their RNA. Then you can't use oligo dT, but you'd need to use random oligos.
Hm, I don't have experience with random oligos....

Minna
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#7 cheerioet

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Posted 30 May 2009 - 12:27 AM

Oh, my answer was not on topic, sorry...
Wait, you said you want to "get fresh phage to extract for RNA", does this mean you're working with bacteria phage RNA? Do you know if that is polyadenylated? AFAIK, bacteria don't polyadenylate their RNA. Then you can't use oligo dT, but you'd need to use random oligos.
Hm, I don't have experience with random oligos....

Minna



Erm...Oh...

Thanks for telling me. I wasn't quite sure. Right now, I am still on the way to get fresh plaque. Will extract them on Monday and use up all the remaining reactions in the left over kit. I hope there will be something, if not; my boss got to kill me :unsure:

Thanks in advance.





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