I am trying to isolate Fosmid DNA from my clones by using a Fosmid purification kit but every time I am getting two bands on the gel so I am not able to give my samples for sequencing. How can I confirm that the second band is from the host genome or from the fosmid that some how was nicked?
I am using fosmid DNA purification kit of epicentre and E.coli strain is EPI 300 that is given along with fosmid library preparation kit.
what is the expected size of your fosmid? It can be only the different forms of the fosmid(coilled, super coilled and broken). Personaly, for big plasmid such as fosmids and cosmids I prefer to do the sambrook method followed by a phenol-chloroform. The main thing is to carrefully pipet and to limit time incubations with lysis buffer. Normaly if you perform your migration on a 0.8% gel you have no problem to differenciate gDNA and fosmid.
I think you are answering other problem...
i wll explain... I am preparing fosmid metagenomic library . After ligation and transformation i am screening for clones of my interest then i have to sent these positive clones for sequencing but when i was isolating the DNA from these clones by using the kit that i had told earlier i am getting two bands one band is of required size (approx 48kb : 40kb insert + 8 kb vector) but other one is above this . i had also done restriction to check whether it is of fosmid or genomic and i got smear as in case of genomic DNA that made me to think that genomic is also getting isolated along with fosmid DNA so i also took a negative control(only host without vector and insert ) and to my surprise no genomic DNA was seen on gel when isolation was done with same kit. This is where i am that if it is not genomic then on restriction why that pattern is obtained....
as u said i also tried isolation by sambrook method but again the results were coming same.
can you suggest me what should i do to confirm that it is genomic or my DNA in fosmid.
without a gel picture, it is difficult to appreciate your problem. Are the bands very different in size? if no, it could be link to a mix of two clones...you think you have one clone, but perhaps you have collected two clones. It can happens.
This problem is not with a single clone it is with most of the clones so i dnt think so that there are two clones every time ....and yes there is difference in size too
Perhaps you could cut out the band of interest to purify it. The large upper band does look consistent with genomic DNA and if your restriction cutting resulted in some known bands from fosmid and a DNA smear then purifying the band from the gel and cutting it without the other band could help you determine if the upper band is indeed genomic. It will tell you if you still get the DNA smear when digesting just the lower purified band. I wonder if is it possible that the fosmid DNA could "drag" some genomic DNA along with it and that is why you don't see the upper band in the no fosmid control preps?
ys absolutely i was also thinking that fosmid DNA is somehow dragging the genomic DNA with it which is not seen in negative ones........i also tried restriction with DNA where i was getting an band in accordance with that of my insert but at the same time there was smear just below that also....i wll separately restrict them and then analyse the result......thnx :)
@Andrija Finka I was not vortexing it and after adding lysis solution i invertd it slowly and then furthr added next solution and then incubated for 5 mins. and then followed the next steps ...do u think there is some problem in this??
Make a time titration 1-5 minute and you will see when your genomic DNA detaches. Be aware that one lot of kit may differ to another.
What do you mean positive clones from your fosmid library? My educated guess is that you mean the one that grow on your drug selection plates? We usually pick about 4 of these and do PCR using the fosmid primers provided with the kit then sequence the PCR products. This procedure will verify the actual sequence on the fosmid. Then we come back to that "clone" and purify the fosmid from it (qiagen plasmid mini kit works OK).
fosmid is circular, right? just clean your sample by using Plasmid safe DNAse. it degrades everything linear and leaves everything circular.
By using the a kit of hihg through put and with 1-5 mins titration your genomic DNA detaches. I have used qiagen plasmid mini kit. U may try it.
Hi
what protocol you followed for sequencing your 40Kb Fosmid insert?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology