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First you need to make sure your PCR works and your gene is expressed in the new cell line. If the answer to both questions is yes, then there is possibility that the two types of cells give you different sized bands, or one does not give you amplification at all due to alternative splicing. Even you don't know what is going on in the two cells, but you can look into databases such as NCBI AceView to know the structure of the gene and potential isoforms. You should also check your primer location to know which region your primers bind to. You can use the in silico PCR tool or the Splice Centerto do so. After gathering all the information, you should predict whether the bands you got are non-specific or isoforms.
#153913 will mRNA from different cell line lead to loss of expected product in RT PCR?
Posted
on 17 April 2013 - 10:02 PM
#153174 PCR with Plasmid recovered from filter paper
Posted
on 01 April 2013 - 04:48 PM
If that is the case, just elute the plasmid with 50ul of TE buffer or less by suspending and centrifuging at full speed if my memory serves me right.
after that just add in your competent cells and do the transformation.
after that just add in your competent cells and do the transformation.
#153090 PCR with Plasmid recovered from filter paper
Posted
on 27 March 2013 - 11:25 PM
you can use 50ul of TE buffer/nuclease free water to elute the plasmid.
It's better to do transformation rather than doing PCR directly. You never how much of plasmid has been loaded into the paper.
Is the plasmid in FTA card? There are instructions on their website on how to elute plasmid from it.
It's better to do transformation rather than doing PCR directly. You never how much of plasmid has been loaded into the paper.
Is the plasmid in FTA card? There are instructions on their website on how to elute plasmid from it.
#152994 PCR with Plasmid recovered from filter paper
Posted
on 26 March 2013 - 11:17 PM
I suspect that the main reason the blog suggested using it only for transformation is that you will then have a glycerol stock of bacteria containing the plasmid that you can go back to time and again to produce more plasmid if and when you need it.
Rather than using it all up for PCR and then having to send for more when you run out. Or contaminating your single aliquot while using it for multiple PCRs etc.
I can't see any reason why you wouldn't be able to use it for PCR. I routinely run QPCR on DNA samples in saliva collected onto filter paper. I actually just put a little punched circle of the paper directly into my master mix and run. There are specific tools available for this purpose, if you need to be doing a high number of these.
If I were you, I would elute into water then use a few ul to transform into bacteria and a few ul for your PCR, then store the rest as a back up until you have verified your transformation was successful and you have isolated and verified the plasmid from your new stock.
Rather than using it all up for PCR and then having to send for more when you run out. Or contaminating your single aliquot while using it for multiple PCRs etc.
I can't see any reason why you wouldn't be able to use it for PCR. I routinely run QPCR on DNA samples in saliva collected onto filter paper. I actually just put a little punched circle of the paper directly into my master mix and run. There are specific tools available for this purpose, if you need to be doing a high number of these.
If I were you, I would elute into water then use a few ul to transform into bacteria and a few ul for your PCR, then store the rest as a back up until you have verified your transformation was successful and you have isolated and verified the plasmid from your new stock.
