HELP No DNA after Nucleic Acid Extraction (Qiagen kit) - (Aug/21/2008 )
QUOTE (smu2 @ Aug 22 2008, 04:57 PM)
QUOTE (gortat @ Aug 22 2008, 07:15 AM)
Thanks a lot.
My solution does turn clear after adding the lysis buffer. I have a high copy plasmid (pET23b). I obtain the same result (no DNA) with mini and midi. Until now I was quite sure that the plasmid is present because I was using a glycerol stock to run my cultures (previously I could obtain decent quantity of DNA using the same tube of glycerol stock). We always prepare two tubes of glycerol stock. One to work with and the other one "untouchable", both kept in different freezers. I tried to extract DNA from the "untouchable" one in case if the working one got thawed and bacterias died. Still nothing.
However I will do PCR just to make sure that the plasmid is present. Thanks a lot.
My solution does turn clear after adding the lysis buffer. I have a high copy plasmid (pET23b). I obtain the same result (no DNA) with mini and midi. Until now I was quite sure that the plasmid is present because I was using a glycerol stock to run my cultures (previously I could obtain decent quantity of DNA using the same tube of glycerol stock). We always prepare two tubes of glycerol stock. One to work with and the other one "untouchable", both kept in different freezers. I tried to extract DNA from the "untouchable" one in case if the working one got thawed and bacterias died. Still nothing.
However I will do PCR just to make sure that the plasmid is present. Thanks a lot.
Hmm...starting to sound more strange being that it was working before and now its not. Have you tried streaking out your glycerol stock onto plates? Are you sure that there isn't DNase contamination somewhere in your lab - are preps from other plasmids turning out ok? Are other people having problems?
Everything grows well on plates (I have tried with different antybiotics, from different suppliers). I have just ordered the DNAse/RNAse free water to prepare my LB medium and use it in my preparation. However, all the institute uses MiliQ water from the same source and only my lab has this problem. Everybody from my lab have the same problem trying to obtain DNA from bacterias bearing different plasmids with different resistance.
-gortat-
QUOTE (gortat @ Aug 22 2008, 08:51 AM)
Everything grows well on plates (I have tried with different antybiotics, from different suppliers). I have just ordered the DNAse/RNAse free water to prepare my LB medium and use it in my preparation. However, all the institute uses MiliQ water from the same source and only my lab has this problem. Everybody from my lab have the same problem trying to obtain DNA from bacterias bearing different plasmids with different resistance.
Sounds like a DNase problem that's affecting everyone. To be certain, you could ask a nearby lab if you could use their stuff for a test run. Take some of your cells and do a miniprep in their lab using their pipettors, solutions, etc and see if it works. I would not worry about the contamination being in the LB. The DNA should actually be protected untiil you lyse the cells. You can rinse them in some DNase-free water before you add buffer 1 if it concerns you. Most likely the contamination is in the pippettors, or your buffers, perhaps in your gel tank. You could get some DNase away, or something equivalent and wash down everything in the lab area where you do your preps - including your bench, gel boxes and microcentrifuge, perhaps take apart your pippettors and autoclave them, make new TBE buffer for running your gels, etc. Tracking down the source would probably be difficult so it would be easier to just suspect everything and start out as clean and fresh as possible. Although, I wouldn't throw away your mini/midi prep solutions since they're expensive. Get it working again and then try the solutions to see if they are ok.
Good luck.
smu
-smu2-
QUOTE (gortat @ Aug 22 2008, 08:51 AM)
QUOTE (smu2 @ Aug 22 2008, 04:57 PM)
QUOTE (gortat @ Aug 22 2008, 07:15 AM)
Thanks a lot.
My solution does turn clear after adding the lysis buffer. I have a high copy plasmid (pET23b). I obtain the same result (no DNA) with mini and midi. Until now I was quite sure that the plasmid is present because I was using a glycerol stock to run my cultures (previously I could obtain decent quantity of DNA using the same tube of glycerol stock). We always prepare two tubes of glycerol stock. One to work with and the other one "untouchable", both kept in different freezers. I tried to extract DNA from the "untouchable" one in case if the working one got thawed and bacterias died. Still nothing.
However I will do PCR just to make sure that the plasmid is present. Thanks a lot.
My solution does turn clear after adding the lysis buffer. I have a high copy plasmid (pET23b). I obtain the same result (no DNA) with mini and midi. Until now I was quite sure that the plasmid is present because I was using a glycerol stock to run my cultures (previously I could obtain decent quantity of DNA using the same tube of glycerol stock). We always prepare two tubes of glycerol stock. One to work with and the other one "untouchable", both kept in different freezers. I tried to extract DNA from the "untouchable" one in case if the working one got thawed and bacterias died. Still nothing.
However I will do PCR just to make sure that the plasmid is present. Thanks a lot.
Hmm...starting to sound more strange being that it was working before and now its not. Have you tried streaking out your glycerol stock onto plates? Are you sure that there isn't DNase contamination somewhere in your lab - are preps from other plasmids turning out ok? Are other people having problems?
Everything grows well on plates (I have tried with different antybiotics, from different suppliers). I have just ordered the DNAse/RNAse free water to prepare my LB medium and use it in my preparation. However, all the institute uses MiliQ water from the same source and only my lab has this problem. Everybody from my lab have the same problem trying to obtain DNA from bacterias bearing different plasmids with different resistance.
I have that before with some of my glycerol stocks. what I did is use fresh antibiotics (high doses) and streak the glycerol on plates and pick few colonies from plates and grow them in LB (high dose Amp) and I was able to get my plasmids back.
good luck
-fadila-
QUOTE (smu2 @ Aug 22 2008, 07:00 PM)
QUOTE (gortat @ Aug 22 2008, 08:51 AM)
Everything grows well on plates (I have tried with different antybiotics, from different suppliers). I have just ordered the DNAse/RNAse free water to prepare my LB medium and use it in my preparation. However, all the institute uses MiliQ water from the same source and only my lab has this problem. Everybody from my lab have the same problem trying to obtain DNA from bacterias bearing different plasmids with different resistance.
Sounds like a DNase problem that's affecting everyone. To be certain, you could ask a nearby lab if you could use their stuff for a test run. Take some of your cells and do a miniprep in their lab using their pipettors, solutions, etc and see if it works. I would not worry about the contamination being in the LB. The DNA should actually be protected untiil you lyse the cells. You can rinse them in some DNase-free water before you add buffer 1 if it concerns you. Most likely the contamination is in the pippettors, or your buffers, perhaps in your gel tank. You could get some DNase away, or something equivalent and wash down everything in the lab area where you do your preps - including your bench, gel boxes and microcentrifuge, perhaps take apart your pippettors and autoclave them, make new TBE buffer for running your gels, etc. Tracking down the source would probably be difficult so it would be easier to just suspect everything and start out as clean and fresh as possible. Although, I wouldn't throw away your mini/midi prep solutions since they're expensive. Get it working again and then try the solutions to see if they are ok.
Good luck.
smu
Actually, I have just tried that (several paralel tests today). If I start from the same glycerol stock using ALL the stuff (including flasks, culture media and their both Mini and Midi kit, still Qiagen) from another lab I have DNA!!! But if I only try to do the bacterial culture in my lab and THEN I do everything in the same neighbour lab...nothing. So, the first case really indicates the DNAse contamination but I still can´t explain the second one since my flasks were sterile and no tips nor pipets were from my lab.
I think that the simplest way is to start from the beginning with completely new buffers after treatment against DNAses.
Thanks smu, I owe you
-gortat-