Miniprep DNA digestion smear - (Mar/16/2008 )
hello everyone
I m trying to make an over expression construct. After ligation of my cDNA full with linker and vector, I transformed E.coli.
After getting plasmids I cut with 2 RE, but I got a long smear, no bands!.
Any one know what is the problem?, it is a DNA degradation?, I used the same vector for knock don and I succeeded!.
Hi,
Which enzymes you are using?
Which enzymes you are using?
I m using SpeI/HindIII for cDNA fll length, and XhoI/HindIII for Linker, and the digestion of miniprep DNA with SpeI/XhoI.
Which enzymes you are using?
I used SpeI/XhoI
Which enzymes you are using?
I used SpeI/XhoI
may this will be helpful? Are you sure there are no other restriction sites in your insert or vector for the enzymes u've used.
I think you should make some controls.
And checking your miniprep. did u run the plasmid after mini prep
I have another restriction site, but I tried another enzymes and I got same smear from well to bottom!.
check what type of cells you transformed into - some e.coli have endonucleases that have to be removed from the DNA prep or else they cause DNA degradation as soon as you try to do anything with your DNA. If you're using a miniprep kit make certain to do the extra wash steps that they recommend.
Thank you for reply
I usually use E.coli DHa5, and I have a result with it. I made a PCR from miniprep DNA and I got the expected band, but when I do miniprep DNA digestion I got smear!
How are you doing the miniprep?
What, exactly, is going into your restriction digest? How much DNA, what volume? How much enzyme, buffer, BSA, water? How long are you incubating?
Does you ladder look good on your gel?
What, exactly, is going into your restriction digest? How much DNA, what volume? How much enzyme, buffer, BSA, water? How long are you incubating?
Does you ladder look good on your gel?
Thank you for reply
I mix 7.5 water+0.5 H buffer+0.5 M buffer + 0.25 SpeI + 0.5 XhoI+ 1ul miniprep DNA. Incubation 37c for 1h.
yes my ladder is good in gel.