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Problem in shRNA Annelaing ( Help Required )


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#1 The Gene

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Posted 20 June 2010 - 07:45 AM

Hi guys .. im trying to anneal lets say top and bottom strand oligos of about 63 bases each. When i run the gel the supposedly annealed band comes at 63 bp, but the single stranded top and bottom oligo strands comes at around 30-35bp.


The single stranded oligo size is 63 bases but when run on the gel it appears at around 30bp. and this is the same scenario for 12 different single stranded oligos i have ordered recently. Kindly help me what u guys suggest.??? and am i clear what i am trying to say?? let me know if any





THNX
THE GENE

#2 stardust

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Posted 21 June 2010 - 05:18 AM

Hi,

this is normal, single stranded oligos run fast than double stranded oligos. The DNA ladder is made from double stranded DNA. After annealing you always get a mixture of single and double strand and only double strand runs at the expected size as your ladder is double stranded DNA as well. Just continue, no problem, I have done this for way over 20 oligos!

Stardust



Hi guys .. im trying to anneal lets say top and bottom strand oligos of about 63 bases each. When i run the gel the supposedly annealed band comes at 63 bp, but the single stranded top and bottom oligo strands comes at around 30-35bp.


The single stranded oligo size is 63 bases but when run on the gel it appears at around 30bp. and this is the same scenario for 12 different single stranded oligos i have ordered recently. Kindly help me what u guys suggest.??? and am i clear what i am trying to say?? let me know if any





THNX
THE GENE



#3 Functional Screens

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Posted 21 June 2010 - 10:33 AM

The (top and bottom) single stranded oligos were "self-annealed", just like shRNA (but it's shDNA).




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