Genomic sequencing - (Nov/02/2005 )
I have a huge problem while trying to sequence some genomic DNA. I am starting from E. coli. I tryed different protocol for DNA extraction (QIAgen or CTAB purification). I have been very careful to remove any trace of ethanol in my final product and resuspend the product in water since the people from the 2 sequencing facilities that tried to sequence my DNA samples told me that it was better than TE or even Tris buffered solution... and I get nothing!
Since I was looking for transposons insertion points I also asked for sequence of a unique gene to be performed (in the unlikely case of multiple insertions), and this does not work either
I am now starting to become a little mad with this experiment, and my boss is giving me a hard time. But I cannot go further without those sequences.
Any suggestions to improve things? Any idea to make sure my DNA is good enough for sequencing (it's starting to become expensive) before I send it to the sequencing facilities?
have you checked your preparation by an agarose gel? how does that look like? did you check uv-vis spectra?
I did not load it on a gel, what should I expect from genomic DNA?
AS for UV I measured concentration in TNE Buffer (7 samples all around 2000µg/ml, dilution made so that readings were below 1OD) and A260/A280 were between 1.9 and 2.1. In the latter case, when Itried further purification I lost almost all my DNA, so I sent it like that...
i'd try to go for a 0.5-0.7% agarose gel. for sure you can check if there are some smaller fragments (dna maybe degraded?)
are you sure, that everything you use is dnase free/sterile? in general purity from quiagen kit should be fine. did you maybe shear the dna by vortexing?
2 mg/ml is an awful large amount of DNA to recover, I'm not sure which procedure you used, but you may want to recheck your calculations here and make sure you are including what you need in the sequencing reaction. As for the sequencing, are you sure the sequencing primers are correct? Are your sequencing reactions completely failing?
Well for the calculation, the spectrophotometer did them for me. But I also cross checked with the reading and made the calculation by my self, it seems OK.
2mg/ml may seems a lot, but for sequencing I am supposed to provide 20µg, and my final volume is only 50µl... So I am just concentrating a lot in the end. The protocol I used is from Current protocols. The only modifications are:
- repeat 2 or 3 times the CTAB step
-add a chloroform/isoamyl alcohol step after the phenol/chloroform/IAA to be sure to get rid of all the phenol.
I never vortexed my preps, only shake them gently when needed. I am trying to see how it looks on agarose too.
Thanks for the help
It sounds like you are trying to sequence directly off E.coli genomic DNA which is in general quite hard (much harder than plasmids or PCR products). Can you post your exact protocol here for us to look at and make suggestions?
Improving DNA sequencing
Yes I am sequencing directly from the genome. I needed to use a genomic marker for my experiments, and I want to know where it is inserted in the genome of my strains.
For the extraction protocol (in brief):
1- digestion of my cells with proteinase K
2- Addition of salts (5M NaCl)
3- Addition of CTAB and 10' incubation at 65ºC
4- Addition of Chloroform/IAA, spin, and save supernatant
5- Redo 3 and 4 until I do not see any the white blob at the interface.
6- Addition of Phenol/Cloroform/IAA spin and save supernatant
7- (i added this step to the original protocol) Addition of Chloroform/IAA, spin, and save supernatant.
8- Precipitation of DNA with isopropanol, wash with 70% Etanhol, dry and resuspend in water.
Since I am giving my samples to other people I have no idea of the protocol for Sequencing
Thanks for your help
I suspect that the problem then is they way the sequencing prep is being done. It is more than likely that they are applying a standard protocol designed for plasmids to your DNA and it is not working very well. Try to find out what they are doing and post it here if you can.
Another alternative is to use a PCR based approach that can amplify flanking DNA such as panhandle PCR. It is pretty easy to get this working on bacterial DNA.
Sequencing DNA protocols
Sorry I don't know.
I have my sequence done at the Robarts Institute, they used the Big dye terminator chemistry. I gave them a 15µl mix containing about 2µg genomic DNA and 1.7µM of my primers. I am not sure this could help...
I also run a gel as suggested Kersten. There is some sheared DNA, but the bulk of it appears as big "potato" high on the lane, so I am rather confident that I did not do too much damage.