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help needed in cDNA synthesis


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#1 cheerioet

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Posted 29 October 2009 - 04:24 AM

Hi,

I did until 2nd strand cDNA synthesis and checked on it after PCR via gel electrophoresis, just to make sure if I got something there...but the gel image came out to have only the ladder.

the material I am using for the starting if a poly-A-tailed rna (using some commercial kits), and then i started with the decapping of the rna with reagents provided from a kits as well.

after that, i continued on with the 1st strand cDNA synthesis using the provided 1st strand primer and removal of the rna from the 1st strand before proceeding to the 2nd strand cDNA synthesis and amplification.

the primers used are also provided in the kits and after the pcr cycles, i view using 1% EtBr stained agarose gel with a 1 kb ladder marker to check if i had some pcr product which is around 4kb.

not my lucky day...i had been trying this and still, i did not manage to get any...

May I know if there is a way to synthesis 2nd strand of the cDNA without using any specific primers? This is because I need the whole length and i did not have the sequence of the interested gene. that's why i need to do a cDNA library cloning.

I hope some1 out here can help to advise or any suggestion or any solution to deal with this?

Thanks in advance.

Cheerioet :)

Edited by cheerioet, 29 October 2009 - 04:25 AM.


#2 susanna

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Posted 10 February 2010 - 08:26 AM

how did you isolated your RNA?

I would just do first strand synthesis, followed by qPCR. You can check the quantity of the cDNA, using nanodrop.

Hi,

I did until 2nd strand cDNA synthesis and checked on it after PCR via gel electrophoresis, just to make sure if I got something there...but the gel image came out to have only the ladder.

the material I am using for the starting if a poly-A-tailed rna (using some commercial kits), and then i started with the decapping of the rna with reagents provided from a kits as well.

after that, i continued on with the 1st strand cDNA synthesis using the provided 1st strand primer and removal of the rna from the 1st strand before proceeding to the 2nd strand cDNA synthesis and amplification.

the primers used are also provided in the kits and after the pcr cycles, i view using 1% EtBr stained agarose gel with a 1 kb ladder marker to check if i had some pcr product which is around 4kb.

not my lucky day...i had been trying this and still, i did not manage to get any...

May I know if there is a way to synthesis 2nd strand of the cDNA without using any specific primers? This is because I need the whole length and i did not have the sequence of the interested gene. that's why i need to do a cDNA library cloning.

I hope some1 out here can help to advise or any suggestion or any solution to deal with this?

Thanks in advance.

Cheerioet :)



#3 cheerioet

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Posted 13 March 2010 - 04:30 AM

how did you isolated your RNA?

I would just do first strand synthesis, followed by qPCR. You can check the quantity of the cDNA, using nanodrop.

Hi,

I did until 2nd strand cDNA synthesis and checked on it after PCR via gel electrophoresis, just to make sure if I got something there...but the gel image came out to have only the ladder.

the material I am using for the starting if a poly-A-tailed rna (using some commercial kits), and then i started with the decapping of the rna with reagents provided from a kits as well.

after that, i continued on with the 1st strand cDNA synthesis using the provided 1st strand primer and removal of the rna from the 1st strand before proceeding to the 2nd strand cDNA synthesis and amplification.

the primers used are also provided in the kits and after the pcr cycles, i view using 1% EtBr stained agarose gel with a 1 kb ladder marker to check if i had some pcr product which is around 4kb.

not my lucky day...i had been trying this and still, i did not manage to get any...

May I know if there is a way to synthesis 2nd strand of the cDNA without using any specific primers? This is because I need the whole length and i did not have the sequence of the interested gene. that's why i need to do a cDNA library cloning.

I hope some1 out here can help to advise or any suggestion or any solution to deal with this?

Thanks in advance.

Cheerioet :lol:


hmm..thanks for the suggestion, but actually to do qPCR isn't my purpose...and I am indeed using nanodrop to check the quantity and quality...yet, no positive result.
hence now, being stopped and trying to use other type of approach...

thanks for your suggestion.

#4 susanna

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Posted 17 March 2010 - 08:18 AM

usually nanodrop is used to check the quantity of your DNA
To check the quality, maybe its better to use gel

how did you isolated your RNA?

I would just do first strand synthesis, followed by qPCR. You can check the quantity of the cDNA, using nanodrop.

Hi,

I did until 2nd strand cDNA synthesis and checked on it after PCR via gel electrophoresis, just to make sure if I got something there...but the gel image came out to have only the ladder.

the material I am using for the starting if a poly-A-tailed rna (using some commercial kits), and then i started with the decapping of the rna with reagents provided from a kits as well.

after that, i continued on with the 1st strand cDNA synthesis using the provided 1st strand primer and removal of the rna from the 1st strand before proceeding to the 2nd strand cDNA synthesis and amplification.

the primers used are also provided in the kits and after the pcr cycles, i view using 1% EtBr stained agarose gel with a 1 kb ladder marker to check if i had some pcr product which is around 4kb.

not my lucky day...i had been trying this and still, i did not manage to get any...

May I know if there is a way to synthesis 2nd strand of the cDNA without using any specific primers? This is because I need the whole length and i did not have the sequence of the interested gene. that's why i need to do a cDNA library cloning.

I hope some1 out here can help to advise or any suggestion or any solution to deal with this?

Thanks in advance.

Cheerioet :D


hmm..thanks for the suggestion, but actually to do qPCR isn't my purpose...and I am indeed using nanodrop to check the quantity and quality...yet, no positive result.
hence now, being stopped and trying to use other type of approach...

thanks for your suggestion.






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