Hey, I am new to molecular biology. I have a plasmid which contains my desired gene and I have amplified the gene using pcr. Now I need to do agarose gel electrophoresis. I have a lambda DNA HindIII marker. If I get to visualize (by UV light) a band in gel on the lane of my DNA sample, may I know the number of bps corresponding to that band? If yes, how to record that as a data?
the band sizes you can see are
564, 2027, 2322, 4361, 6557, 9416, 23130 bp
To approximately size your product either view it between 2 lambda bands and estimate its size or measure the distances S that the band have travelled through the gel then plot 1/S against band size. Then read off the distance your band has moved against that graph will give you an approximate size for the fragment, The graph will be quite linear. The further you run the gel the more accurate this will be. It is quite wide range and many people use a 100 bp ladder which gives great accuracy from 0 to 1500 bases. Absolute accuracy is usually not needed for pcr as if you have only one band of approximately the right size it is almost always the right amplimer
You can deduce the approximate size of your PCR amplified product from the agarose gel, by comparing your amplicon with that of the standard maker lane. If you would like to know the exact bases that you amplified, you could sequence your PCR product using the primer pair you used to amplify to start with. From that result, you could order more sequencing reactions to cover the whole sequence.
Hope this helps!
All the best,
Manjunatha
the band sizes you can see are
564, 2027, 2322, 4361, 6557, 9416, 23130 bp
To approximately size your product either view it between 2 lambda bands and estimate its size or measure the distances S that the band have travelled through the gel then plot 1/S against band size. Then read off the distance your band has moved against that graph will give you an approximate size for the fragment, The graph will be quite linear. The further you run the gel the more accurate this will be. It is quite wide range and many people use a 100 bp ladder which gives great accuracy from 0 to 1500 bases. Absolute accuracy is usually not needed for pcr as if you have only one band of approximately the right size it is almost always the right amplimer
You won't know the exact base number of your gene-of-interest by just running a gel. DNA size markers only show you an approximate size of your gene. You will have to sequence it (the PCR product amplified by specific primers covering the beginning and end of the gene) and count the bases from the sequenced DNA profile to find out.
To know the exact base pair of your product you need to sequence it but for approximation you can use Alpha view after taking your gel picture from gel documentation system. hope it helps
Hi Rajdeep.
If your primers are specific and good they would amplify one product even from complex templates (genomic DNA or other mixture of DNAs). If you are amplifying from a clone as you suggested you should not amplify anything but your gene of interest.You simply need to compare the size of your amplicon with the sizes of the Lambda HindIII digest to get an idea about its approximate size. Most of the time you know the size of your expected product and if that matches with our result then you are sure that you have amplified the right product. To be certain that there has been no mutation, deletion etc you will have to sequence your product, really depends on what your downstream applications are.
Anindita
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