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Troubleshooting qPCR standards

qpcr rtpcr pcr cloning

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#1 janaki

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Posted 02 July 2014 - 07:38 AM

I've been working on generating qPCR standards in plasmid form. I have used the pCR4.1 Topo-TA system and verified a few clones using PCR for the presence of my insert (the two fragments I've cloned are 200 bp and 110 bp), the bands are nice and bright and correctly sized. 

When I do a test qPCR run using SYBR green, I have no amplification. A positive control set of standards which used another template was successfully amplified. I also tried cleaning up the plasmids using precipitation to no avail. I have attached the conventional PCR, there are four clones for each insert and two negative controls.

Any idea what I have overlooked? Thanks to anyone who can help!

 

2X SYBR green

10 umoles each primer
in 20 ul reaction

95 2 min
95 30s 
55 30s
72 30s 

 

45 cycles

Attached Thumbnails

  • conventional pcr gel.jpg

Edited by janaki, 02 July 2014 - 07:41 AM.


#2 Trof

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Posted 02 July 2014 - 12:22 PM

How much of the plasmid did you put into reaction?

Plasmids are hugely efficient templates and only require pg amounts (depending on the size even less).

Too much template inhibits PCR reaction. SYBR reactions are even more sensitive to inhibition, since SYBR itself is a mild inhibitor.

My first guess would be too much plasmid in the reaction.

 

Second, supercoiled variants of plasmids are on the contrary sometimes difficult templates.

For the creation od qPCR standard, plasmids are usually cut with a single cutter (opposite of target fragment) the and ideally buffered with a "dummy" nucleic acid to match the complexity of the real template.(I did that once, but would need to look up what kind of commercial dummy NA I used)


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