no bands after pcr!!!! - (May/25/2015 )

this is the first time i've done PCR and after pcr i ran the DNA on 0.8% agarose without EtBr but i did not get any bands. the concentrations i got were 38.6 and 19.2 for my samples and the nanodrop 260/280 ratios were 2.32 and 1.96 respectively. the concentrations before doing PCR and pcr product purification with sigma-aldrich PCR clean-up kit were 284.7 <260/280= 1.40> and 227.7 <260/280= 1.44> following are the pcr mix concentrations (50uL) buffer; 8.44, MgCl2; 8.44, dNTPs; 6.75, forward and reverse primers; 1uL each, template; 1uL, Taq polymerase; 0.67 uL and temperature settings: initial; 94

ͦ c, 94*c, annealing; 55*c, extention: 72*c, final extention: 72*c, hold at 4*c.

kindly help me out with this. what might be the reason that i m not getting bands after pcr and purification??

1) Try taking a new set of PCR components (PCR buffer, dNTP) and try.

2) Repeated freeze thawing of the PCR components including the primers maybe a reason. Try making aliquots and store them at 4°C

3) Did your positive and negative control worked?

4) Try running a gradient PCR to check for the correct annealing temp.

Hope this helps.

You wrote "...i ran the DNA on 0.8% agarose without EtBr...". Do you mean you forgot the staining or stained the gel afterwards? If the first is true, then it the mistake is obvious, if the second is true, Mad Researcher's topics are good starting points, esp. no 3.

What are the units of all the numbers?

hobglobin on Tue May 26 15:42:49 2015 said:

You wrote "...i ran the DNA on 0.8% agarose without EtBr...". Do you mean you forgot the staining or stained the gel afterwards? If the first is true, then it the mistake is obvious, if the second is true, Mad Researcher's topics are good starting points, esp. no 3.

What are the units of all the numbers?

dhanashree prabhu on Mon May 25 11:57:07 2015 said:

this is the first time i've done PCR and after pcr i ran the DNA on 0.8% agarose without EtBr but i did not get any bands. the concentrations i got were 38.6 and 19.2 for my samples and the nanodrop 260/280 ratios were 2.32 and 1.96 respectively. the concentrations before doing PCR and pcr product purification with sigma-aldrich PCR clean-up kit were 284.7 <260/280= 1.40> and 227.7 <260/280= 1.44> following are the pcr mix concentrations (50uL) buffer; 8.44, MgCl2; 8.44, dNTPs; 6.75, forward and reverse primers; 1uL each, template; 1uL, Taq polymerase; 0.67 uL and temperature settings: initial; 94

ͦ c, 94*c, annealing; 55*c, extention: 72*c, final extention: 72*c, hold at 4*c.

kindly help me out with this. what might be the reason that i m not getting bands after pcr and purification??

 

I seem to have overlooked that point "Without EtBr". Thanks "hobglobin" for pointing it out.

That could be the major reason for not getting any results.

A positive control sample is really important in this case since it is your first time doing PCR. There are just too many variables to reliably pin point what the problem is (assuming the lack of EtBr was a typo or something).

A positive control tells us that your pipetting is good, your PCR machine is working, all reagents are good, etc, and that the problem is unique to your particular sample in question.

"In science, your two best friends will always be your positive and negative controls."