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We have a gene source from a certain type of bacteria, cells were cultured in nutrient broth medium at 60 C for certain time to be used for DNA extraction. Interested gene was obtained by PCR107 using the forward primer with NdeI recognition site (......) and the reverse primer EcoRI recognition site (.....). The amplified fragment with a size of 807 bp was ligated into pTZ57R/T vector and sequenced using M13/pUC forward and112 M13/pUC reverse sequencing primers. Recombinant plasmid was digested with endonucleases NdeI and EcoRI and the fragment containing the lipase gene was ligated into the expression vector pET-26b (+) which had been digested with the same enzymes. Finally, the ligated plasmid was transformed into E. coli BL21. Isnt it enough to use only pTZ57R/T vector for both purposes? I would be glad to get some fresh information.
Thanks in advance
I've seen this Invitrogen system "E-Gel Clonewell2" that promises you can extract your desired sized band WITHOUT gel extraction.
This will be very useful if works, because I usually lose a lot of DNA during gel extraction.
Has anyone used this and had good/bad experience?
1) When I tried to clone the c-KIT gene into a mammalian expression vector (pCDNA3.1) or viral vector pBABEpuro, I always ended up with big and small colonies on the LB+Amp plate. Only the small colonies were proved to be potentially correct.
2)Then it comes with the second issue, sometimes I have to wait for 2 days to see those small colonies to show up, by then random star colonies showed up too. This makes it very hard to pick the right colonies. Many times I had to pick a dozen of them to get a single correct colony.
3)Third, this is not the end of the story but the beginning. Once I mini-prep a correct colony and enzymatic digestion confirmed it, I scale up for a maxiprep. I ususally inoculate 20 microliters of the mini culture into a big flask contain 250ml LB plus 100ug/ml ampcilin for overnight or I just inoculate 5ml of the culture into a big flask and collect the culture several hours later. The maxiprep ends up with two typical outcomes: either extremely low yield (around 10ug total from 250ml), or normal yield (up to 1mg) but the DNA show a additional major band on gel (~2.2k bp). I can sequence the insert and see correct result but I am very suspicous about the additional band.
4) In many cases, when the maxiprep gives normal yield, I often found stop codon mutation in my gene and no additional band on gel.
5) I tried different vectors and bacteria strains, similar phenomena was observed
6) When I cloned other genes, everything was fine.
7) When I cloned certain mutant c-kit, there was no problem.
Based on above observations, it seems that the c-kit gene is toxic to the bacteria but there shouldn't be any expression in bacteria with the mamalian expresison vector right? I even tried to put small molecule inhibitors for c-kit protein in the bacterial culture which did not work around the problem either.
After electrophoretic migration of my PCR products, I cut my gel band. Then, I have purified my DNA using the Qiaquick gel extraction kit. After nanodrop, I obtained a correct DNA yield (about 20ng/ul ), but I had also an absorption pick at 230nm (A260/230=0.02). I don't know what is it. Agarose or co-purified molecule from the kit buffer?
I'm confused because I want to use these purified products for cloning. Is someone experinced similar problem? Do you think that the cloning may work with abosrbance ratio?
I have a low copy plasmid (ori=pBR322) and I wanted change it to high copy (like ori=pUC).
This paper seemed to say that only one mutation was required to increase the copy number:
However, I made this mutation in my plasmid and it didn't change the copy number. My plasmid does not appear to have rop either.
What are the key mutations between pBR322 and pUC that cause pUC to have such a high copy number?
I am planning to do functional analyses for a novel mutation to investigate its effect using cell based cloning. The data from literature indicate that pET+25b is the best vector to create the expression construct. My target gene is MUTYH (ENST00000450313), which spans for about 1888 bp. However, I do not know what is the most suitable restriction enzyme to do the digestion.
Thanks in advance
I have been trying to clone a genomic region (Promoter + InEx) that sums up close to 10kb, so far it has been unsuccessful with restriction based cloning and have opted for a gateway strategy which now too has become a headache. I am amplifying my fragment with attB1 and attB2 sites added to my primers and afterwards proceed to purifying from the gel. I have also done the respective calculations to leave the reaction equimolar with the pDONR201 plasmid and left incubating the reaction overnight (up to 18 hours). Afterwards I transform DH10B E. coli and end up with 1 or 2 colonies growing on kanamycin plates if any at all and only after a long incubation!!!! I have also tried a Carbenicillin resistant version of pDONR221 and have only gotten empty vectors which in principle should not happen since there is also de presence of ccdB within the vector?? I have also tested different BP Clonase II batches and have ruled out the enzyme mix.
If anyone has any experience in cloning this size fragment I or had to deal with any of these issues before I would appreciate any input!
What is the best way to get good yield reproducibly? This is a stupid thing to get held up on, and it seems to be our Achilles heel. We have tried kits from Epoch, QIAgen and the old school freeze and squeeze, and none of them work well. Any tips, suggestions or ideas?