Ligation problem - (Jun/12/2010 )
Hi!!
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk 
sjk on Jun 12 2010, 05:19 PM said:
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk
i've always used a spectrophotometer
molstudent on Jun 13 2010, 06:57 PM said:
sjk on Jun 12 2010, 05:19 PM said:
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk
i've always used a spectrophotometer
sjk on Jun 14 2010, 11:32 AM said:
molstudent on Jun 13 2010, 06:57 PM said:
sjk on Jun 12 2010, 05:19 PM said:
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk
i've always used a spectrophotometer
Thanks, will try it.
sjk on Jun 14 2010, 09:34 AM said:
sjk on Jun 14 2010, 11:32 AM said:
molstudent on Jun 13 2010, 06:57 PM said:
sjk on Jun 12 2010, 05:19 PM said:
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk
i've always used a spectrophotometer
Thanks, will try it.
i forgot to add even though the concentration may not be exact due to impurities, when doing 1:3 vector to insert ratios it was never a problem since there was always more insert than vector.
sjk on Jun 12 2010, 08:19 PM said:
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk
A few questions -- are you sure you want 1:10 (insert:plasmid)? Usually you want more insert than plasmid... Also, you also know that these ratios are on a molar basis, right?
But, my big question is -- how did you conclude that it's your ratio of insert to vector that's causing your cloning to fail? Most times, for the past twenty years, my lab doesn't even bother to calculate these ratios, we just "go for it", and more times than not, we have successful clones on the first shot. If I had a ligation fail, the insert to vector ratio would be the last thing I'd worry about -- there are so many other possible failure points that are much more likely to be causing your troubles...
HELLO, I AM NEW TO THE SITE AND VERY EXCITED TO ADD AND LEARN AT THE SAME TIME. FOR YOUR PROBLEM WITH LIGATION, I AGREE WITH THE OTHER COLLEAGUES I'VE TRYED TO USE AS MUCH INSERT AS POSSIBLE WHENEVER I DON'T GET ANY CLONES. FOR INSTANCE, IF YOU USE 1 MICROLITER (USUALLY YOU SHOULD PAY ATTENTION TO THE UNITS AS WELL) OF T4 LIGASE, 2 MICROLITER OF BUFFER, 1 MICROGRAM OF PLASMID AND 16 MICROLITERS OF DNA!!!!!!! YOU SHOULD HAVE NO PROBLEMS. I'VE TRYED THIS WITH AN INSERT LARGER THAN 1 kb. I HOPE YOU GET IT, I KNOW WHAT IS TO HAVE THIS PROBLEM, GOOD LUCK!!!!
Well, that was the ratio that my advisor has found out and it is tested. But its not the ratio but the method to determine the amount of DNA in the samples purified and restriction digested. We get varying concentrations after purification because inevitable loss during the process. Well thanks for the suggestions will certainly try them out.
HomeBrew on Jun 14 2010, 04:16 PM said:
sjk on Jun 12 2010, 08:19 PM said:
I am having a problem with ligation. It has been weeks since I am trying this. I have used Bam HI and Xho I to digest my insert and Plasmid. The insert was amplified correctly (have checked this using the gel and necessary markers). The insert and plasmid both are purified properly. I am not able to determine the amount of the insert and plasmid to be used. I am using agarose gel and UV for this purpose. I need a ratio of 1:10 (insert:plasmid). Can anyone plz suggest some other method to determine the concentration of DNA in sample?
Thanks,
sjk
A few questions -- are you sure you want 1:10 (insert:plasmid)? Usually you want more insert than plasmid... Also, you also know that these ratios are on a molar basis, right?
But, my big question is -- how did you conclude that it's your ratio of insert to vector that's causing your cloning to fail? Most times, for the past twenty years, my lab doesn't even bother to calculate these ratios, we just "go for it", and more times than not, we have successful clones on the first shot. If I had a ligation fail, the insert to vector ratio would be the last thing I'd worry about -- there are so many other possible failure points that are much more likely to be causing your troubles...
If you have no machine to estimate DNA concentration (a spectrophotometer or a nanodrop), you can estimate your concentration on a molar basis with ethidium bromide only. The best way to do this is to prepare a set of serially diluted standards of known concentration using salmon sperm DNA or calf thymus DNA. Dot your standards on a piece of parafilm, then dot your unknown DNA (vector and insert) in separate dots on the same piece of parafilm. Add some ethidium bromide solution to each dot. Expose the samples to UV and shoot a picture. You should be able to estimate the concentration of your samples by comparing the intensity of the florescence to that of your known concentration controls.
It's been many years since I've done this -- I seem to recall using parafilm, but I'm unsure if UV penetrates parafilm on a transilluminator -- I may be remembering it incorrectly -- maybe it was saran wrap? Or use a hand-held UV source and illuminate from above? Google "ethidium bromide dot quantitation"; you should find something.
Thanks a lot will definitely try searching it. I yesterday estimated concentration using the gel doc systems band analysis menu. Lets c what happens.
HomeBrew on Jun 15 2010, 09:47 PM said:
It's been many years since I've done this -- I seem to recall using parafilm, but I'm unsure if UV penetrates parafilm on a transilluminator -- I may be remembering it incorrectly -- maybe it was saran wrap? Or use a hand-held UV source and illuminate from above? Google "ethidium bromide dot quantitation"; you should find something.