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Picking multiple clones for sequencing two copies of the same promoter - (Dec/05/2013 )

Hi Guys

 

Wondering if you could help me again.  

 

I'm trying to analyse the methylation state of my transgenic promoter in my rice plants and there are two copies of the transgene in the rice line that I'm working on. I've bisulfite-treated my DNA, cloned the PCR fragments and picked 10 colonies for sequencing. So I am wondering:  

 

1) I've been trying to find out why you pick multiple clones and it seems to be because of incomplete conversion. Is this correct? If so then:

 

2) In my initial results there is sequence variation between the different clones but as these are clones from the same bisulfite-treated DNA and the same PCR can I assume that any differences are due to the differences in the methylation state of the two promoters? Unfortunately (despite a number of attempts) we haven't been able to find out where the two promoters are inserted into the rice genome, so I have no way of distinguising between them.

 

Thanks in advance,

 

Kate      

-Kate Warner-

So, you have tried inverse pcr to identify the insertion location?

-phage434-

I have tried amplifying between the constructs (as southerns suggest they are close together) and I've also tried a TAIL approach specifically designed for rice but I could never get it to work.

-Kate Warner-

Inverse PCR is worth trying. Design two primers facing away from each other (toward the genome) on the transposon.

Take genomic DNA and cut with a frequent cutter, such as Sau3AI or other 4 bp recognition site cutter. Make sure there is no site for the cutter between your primer and the end of the transposon.

Heat kill or purify your DNA to get rid of the enzyme.

Work with a relatively dilute sample (you want intramolecular ligation) and ligate the DNA. Somewhere around 20 ng/ul is about right.

Now, use this sample for PCR with your outward facing primers. If things are working you should end up with two bands -- one for each of your transposon insertion sites. You can sequence these fragments to identify the genomic region flanking the transposons. You'll have to gel purify the bands individually.

-phage434-