help for cDNA synthesis - plus RE problem. (Jun/04/2009 )
I have questions over here which i needed people to help me...really desperate for one.
I have a genome sequence for phage. I needed to find suitable RE to cut it. The RE digestion will be done to the phage cDNA.
I am doing 1st strand cDNA synthesis for the phage RNA because we need to keep it stable. The 1st strand, fro my understanding, is just the complementary strand of the RNA to DNA...and it will not be double stranded, instead it will still have rna-dna bond. Protocols suggested to use RNase H to digest it, but the kits I am using allow to use the 1st strand directly for PCR without RNase H treatment.
Am I correct to say that actually after the 1st strand cDNA synthesis, is just a single stranded DNA (because it only involve RT step, not involving PCR). If I needed to get double stranded cDNA, i will need to amplified it?
How am I going to amplified it, if i do not have gene specific primers, and oligo dT isn't suitable. And I tried with random primer, it did not gave me any yield.
I am lost to troubleshoot it, as I a newbie in this field. I would need suggestion and opinion from those who had been involving with this kind of methods.
Can I just directly use the synthesized 1st strand cDNA for RE digestion?
How am I going to find the suitable RE for the phage cDNA? Convert it to complement, then use some programme to get the suitable RE? or i can directly use the phage genome i got from NCBI and put it in the programme to find the RE?
Thanks in advance for the suggestion n opinion.
There you are again...
Let's see, you have phage genome sequence and want to find RE sites to cut it?
http://tools.neb.com/NEBcutter2/index.php (this finds you ORFs and unique RE sites).
"If I needed to get double stranded cDNA, i will need to amplified it?" After RT, you have single-stranded DNA. You need to synthesize the second strand as well.
"I tried with random primer, it did not gave me any yield." Are you confident with your random primer? Did you get good result from a positive control, other mRNA for example? I don't have much experience with them, but I guess finding a good annealing temperature might be a challenge.
"Can I just directly use the synthesized 1st strand cDNA for RE digestion?" No. RE cut only double-stranded DNA.
If I were you, I would try to get the random oligos to work, maybe try different temperatures. Then use randoms for 2nd strand and clone cDNA into a TOPO TA vector. Then you have some RE sites at your genes and can continue with whatever....
Hope this helps,
Cheers,
Minna
Minna on Jun 4 2009, 06:04 PM said:
Let's see, you have phage genome sequence and want to find RE sites to cut it?
http://tools.neb.com/NEBcutter2/index.php (this finds you ORFs and unique RE sites).
"If I needed to get double stranded cDNA, i will need to amplified it?" After RT, you have single-stranded DNA. You need to synthesize the second strand as well.
"I tried with random primer, it did not gave me any yield." Are you confident with your random primer? Did you get good result from a positive control, other mRNA for example? I don't have much experience with them, but I guess finding a good annealing temperature might be a challenge.
"Can I just directly use the synthesized 1st strand cDNA for RE digestion?" No. RE cut only double-stranded DNA.
If I were you, I would try to get the random oligos to work, maybe try different temperatures. Then use randoms for 2nd strand and clone cDNA into a TOPO TA vector. Then you have some RE sites at your genes and can continue with whatever....
Hope this helps,
Cheers,
Minna
Wow...what a fast reply I have. Thank god you are here.
The random primer that I am using is provided by the kits as well. If that's the case, I will have to optimise the temperature, instead of using the suggested temperature. Trying to save up the random primer, because of running low, but I guess, I have no choice.
My supervisor suggested me to use pBluescript instead of TOPO....for the cloning.
Now even my plasmid shown some weird band when I had done the digestion on it. The pBluescript suppose to be around 3kb+ and after I done a single RE digestion to it, it seems to land on more than 3kb +, around 5kb+. I find it very weird as well...how to explain that?
I will try to check the possible RE for the phage and will try to do the PCR amplification on the 1st strand cDNA with different temperature tomorrow.
Thanks Minna for your suggestion.
Cheers,
Cheerioet.
Erm...
I needed a help here...
May I know any 2nd strand cDNA synthesis kits that is/are good in the market (fast, easy and reliable). or any 1 step cDNA synthesis kit?
I am trying to look for one to try out...
Thanks.
Cheers,
Cheerioet