maxiprep troubleshoot - (Feb/12/2007 )
I tried different commercial kits (sigma qiagen, genscript) and with all same results.
Plasmid is there but in very low concentration.
other construicts that I worked in parallel are ok.. yield is ok, but with this particular one (15000 kb) seems that its spit out of cells if overgrown. Today i did maxi from 250 ml OD 0.45 and pellet.. again .. pooor.
dont know what happen. when i got my clone i could do minis normally (although with phenol chloroform) and now.. blaaa. some times i get apparently contamination of a smaller plasmid.
if anyone has sugestions... i would appreciate it..
thanks
ps.. ampi ok, LB media, strains.. just getting transformants with jm109. some slow growing xliblue and non for top10, dh5alpha, dam negative NEB (commercial).
Try to increase the antibiotic concentration, you can double it for an overnight culture. Expecially, ampicillin degrades very quickly. Also, you can try to grow the cells at 30C instead of 37 degrees. and dont let the OD of the culture go too hight, harvest it before it goes to O.D of 1.
Thanks Makosad..
I ll try this competent stbl2 cells that precisely have to be grown at 30. I hope I get any transformants. anyway.. i am trrying what u suggested
Thanks
I tried different commercial kits (sigma qiagen, genscript) and with all same results.
Plasmid is there but in very low concentration.
other construicts that I worked in parallel are ok.. yield is ok, but with this particular one (15000 kb) seems that its spit out of cells if overgrown. Today i did maxi from 250 ml OD 0.45 and pellet.. again .. pooor.
dont know what happen. when i got my clone i could do minis normally (although with phenol chloroform) and now.. blaaa. some times i get apparently contamination of a smaller plasmid.
if anyone has sugestions... i would appreciate it..
thanks
ps.. ampi ok, LB media, strains.. just getting transformants with jm109. some slow growing xliblue and non for top10, dh5alpha, dam negative NEB (commercial).
Try to increase the antibiotic concentration, you can double it for an overnight culture. Expecially, ampicillin degrades very quickly. Also, you can try to grow the cells at 30C instead of 37 degrees. and dont let the OD of the culture go too hight, harvest it before it goes to O.D of 1.
In my earlier times in the lab I had the same problems.
do not overgrow the culture (12h is enough)
elute a second time
special trick: elute with elution buffer preheated to 50°C
do you use enough lysis and precipitation buffer?
aside from adding more antibiotics, another thing you can do is grow your cells at a reduced temperature. Try 30 celsius. Lowering the growth temperature sometimes helps with plasmid with poor yields.
Also there is that Chloramphenicol trick, where at late/mid log phase you add the antibiotic, inhibiting bacteria growth, but plasmid replication continues.
And finally 250ml isn't alot. There are some bac used in my lab, which requires total culture of 6 liters to yield a decent quantity of DNA.
Thanks Mojul and perneseblue.
from 2 midis (170 ml culture each) I got 150 ug, which looks better than what I was getting. it helped ur suggestions on not overgrowing the culkture (10 hr od less than 1) and growing at 30 C
I tried chloramfenicol but someone told me that might lead to incorporation of RNA nucleotides ( not deoxynucleatides) which is not favorable for ES electroporation.
but for transfection of other cells some people in my lab have good results with this enrichment.
Thanks!
btw, increasing lysis and precipitation buffer also helped!
from 2 midis (170 ml culture each) I got 150 ug, which looks better than what I was getting. it helped ur suggestions on not overgrowing the culkture (10 hr od less than 1) and growing at 30 C
I tried chloramfenicol but someone told me that might lead to incorporation of RNA nucleotides ( not deoxynucleatides) which is not favorable for ES electroporation.
but for transfection of other cells some people in my lab have good results with this enrichment.
Thanks!
btw, increasing lysis and precipitation buffer also helped!
I just got this problem right now. Finished my targeting vector and am trying to get decent amount of DNA for a final sequencing and ES electroporation... works fine in mini, but not midi and maxi. sometimes, look like been contamintated... really sad...