Gel electrophoresis plz help

[Gamewizard]

Hi all,

 

I really need help on this. I recently did a experiment where i ran a gel electrophoresis, and then got a printed copy of the gel image, I had 7 lanes/samples but unfortunately it did not come out all well due to problems with the gel.

lane 1 had 1kbp ladder

lane 2 had undigested plasmid

lane 3 had Ndel digested plasmid

lane 4 had hindIII digested plasmid

lane 5 had Ndel and hindIII digested plasmid

lane 6 had PCR product

lane 7 had 100bp ladder

 

So i can only see the first lane i think and last lane/band patterns, but not all of them.

 

How would I prepare standard curves and use them to calculate the sizes of uncut plasmid, single digest, double digest, and PCR product?

 

please help anyone

[rasing02]

If you can only see the marker i.e your 1st and last lane and not the digest and PCR product that means that nothing is wrong with the gel, something went wrong with your digest and PCR, seems like you dont have DNA in those tubes?? Looks like you did not add dna in the digest or PCR? sometimes that happens...when you are doing a reaction make sure you write it on a piece of paper and tick of every little component you add in ths tube...I hope I helped you. if you are sure you have added DNA then i dont know what the problem might be

 

 

 

 

 

 

quote name='Gamewizard' timestamp='1291484085' post='576000']

Hi all,

 

I really need help on this. I recently did a experiment where i ran a gel electrophoresis, and then got a printed copy of the gel image, I had 7 lanes/samples but unfortunately it did not come out all well due to problems with the gel.

lane 1 had 1kbp ladder

lane 2 had undigested plasmid

lane 3 had Ndel digested plasmid

lane 4 had hindIII digested plasmid

lane 5 had Ndel and hindIII digested plasmid

lane 6 had PCR product

lane 7 had 100bp ladder

 

So i can only see the first lane i think and last lane/band patterns, but not all of them.

 

How would I prepare standard curves and use them to calculate the sizes of uncut plasmid, single digest, double digest, and PCR product?

 

please help anyone

[CharonY]

Standard curve: plot size and migration length on semi-log paper. You cannot measure size with undigested plasmid with this (think about why and how it is different from the other products).

 

If you can see the markers but nothing else you likely made an error somewhere, as mentioned above.

[Gamewizard]

Standard curve: plot size and migration length on semi-log paper. You cannot measure size with undigested plasmid with this (think about why and how it is different from the other products).

 

If you can see the markers but nothing else you likely made an error somewhere, as mentioned above.

 

By that you mean the kbp marker and 100 bp marker fragments dont you ? because I know the size of thier fragments and can measure the distance and plot it on semi-log paper. But how do I get the size of the plasmid products? (uncut, Ndel, HindIII,)

[CharonY]

You cannot get the size accurately of uncut plasmids (at best you can approximate using other plasmids with known size). Now you got a semi-log of size over migration length of linear DNA fragments. How would you use that to read out the size?

Tip: use the only remaining other parameter.

[Gamewizard]

You cannot get the size accurately of uncut plasmids (at best you can approximate using other plasmids with known size). Now you got a semi-log of size over migration length of linear DNA fragments. How would you use that to read out the size?

Tip: use the only remaining other parameter.

 

I have two markers each withh diffrent bands so would I plot them on the same paper? (semilog)

 

I am not sure how to read the size, but once i have done the graph i will try to figure out and get back to you