problems with yields from qiagen spin mini prep kit - (Jan/25/2007 )
if your plasmid is large (more than 7kb), you should pre-heat the elution buffer to increase the yield
How old are the anitbiotics which you are using? Any chance they are degrading.
If so, the satellite bacteria might outgrow the actual bacteria and the DNA yield will be low.
Hi, I had the same problem and in the end gave up on Kits altogether.
I use a manual method using the Solution Formulations for P1, P2and P3 from the Qiagen manual with an Iso-propanol precipitation and routinely get buckets of DNA eg 250ug.
It takes about the same time as the spin columns and is much more satisfying!
I use a manual method using the Solution Formulations for P1, P2and P3 from the Qiagen manual with an Iso-propanol precipitation and routinely get buckets of DNA eg 250ug.
It takes about the same time as the spin columns and is much more satisfying!
I agree one could get loads of DNA in this quasi prep. We use it in our lab too but the DNA is not too clean for sequencing. Therefore we have to use columns and kits for preparing DNA for sequencing.
TOP10 is an E. coli strain, and says nothing whatsoever about what kind of plasmid is or is not present in the cell. A critical issue for plasmid yield is the origin of replication and copy control mechanism of the plasmid backbone. pUC18 plasmids, for example, are high copy number (200-500), while pBR322 plasmids have a relatively low copy number (20-30). F plasmids have single copies in cells.
sorry phage meant to type pCR topo 2.1 not TOP10 as far as i know these have a pUC origin.
thanks for all the suggestions im going to be kept very busy trying them all out!
my plasmid is pCR topo 2.1 which is 3.9kb and my insert would bring it just over 4kb so i doubt it needs this step. thanks
I don't think that the problem is the plasmid (I had work with pCR topo 2.1 and never had problems) for me the problem is that you have to much dna and debris that are blocking the membrane can try diluting more the sample and centrifuge it in several steps, use 2 columns per sample or try a midi.
I use a manual method using the Solution Formulations for P1, P2and P3 from the Qiagen manual with an Iso-propanol precipitation and routinely get buckets of DNA eg 250ug.
It takes about the same time as the spin columns and is much more satisfying!
I agree one could get loads of DNA in this quasi prep. We use it in our lab too but the DNA is not too clean for sequencing. Therefore we have to use columns and kits for preparing DNA for sequencing.
Hi Scolix. I know that the percieved wisdom is that you cant sequence from DNA made by this method. I made some minipreps this way and about 3 weeks down the line needed to sequence them - forgot how I had made them , sent them off and got really clean long sequences back!!!!
I do this all the time now and have never had a failure.
I use a manual method using the Solution Formulations for P1, P2and P3 from the Qiagen manual with an Iso-propanol precipitation and routinely get buckets of DNA eg 250ug.
It takes about the same time as the spin columns and is much more satisfying!
I agree one could get loads of DNA in this quasi prep. We use it in our lab too but the DNA is not too clean for sequencing. Therefore we have to use columns and kits for preparing DNA for sequencing.
Hi Scolix. I know that the percieved wisdom is that you cant sequence from DNA made by this method. I made some minipreps this way and about 3 weeks down the line needed to sequence them - forgot how I had made them , sent them off and got really clean long sequences back!!!!
I do this all the time now and have never had a failure.
Ok, will try next time. Hopefully this works as you say, then I dont have to bother with the kits.
thanks