cloning

[dwinglerspew]

can anyone tell me what are the problems associated with longer cloned seqence?How will you go upon investigating your hypothesis?
this is an assignment qestion and i couldnt find the answer

[protold]

Hola, Today to clone big genes the way is amplify them with PCR from the whole DNA of the organism which carry it. First we design two oligonucleotides wich hybridize with the 5´and 3´ends and carry restriction endonucleases sites to introduce the gene in a subcloning plasmid and after production purification and digestion introduce the gene in an expression plasmid to produce the codified protein.Two are the more frequent problems with long genes, one wich is difficult to find unique sites of cloning wich don´t broke your gene at the subcloning time(As more long more probability to find cut sequences) and second as much longer is the gene more probability of mutations introduced by PCR enzyme. The first problem is overpassed studiying the sequence and preparing the subcloning with rare enzymes, because the commun ones will cut the gene. and the second is solved sequencing the PCR amplified sequence, compare with the original (there are infotmatic tools to do it)and see if the mutations produce changes in the frame (horrible you can´t follow this way) or the mutatios are silent, they only change one neutral aminoacid and the change dont have any risk(gly x val), but if the change means the change of a neutral aminoacid by a charged one (glyx pro) it will affect to the protein conformation. Probably there are more aspects but for me these are more relevants, buena suerte

[JackBean]

| really don't think, that Gly <-> Val mutation would be OK. Gly is often in turns, because if its structure. Also, Pro is not charged.

[protold]

Hola, I´m sorry Jack Bean probably the exemple isn´t the best, I only wanted to say that there are silencious mutations. Excuse me, and have a good day

[JackBean]

yeah, sure.
But I think, that silence mutation is something different. I think it is, when you change DNA sequence, but it still codes for the same amino acid (due to the degeneration of the genetic code)

[kk]

Hello, I would like to insert an enhancer element into a cis-reporter plasmid (luciferase) to test promoter activity of my gene of insert. I found the consensus enhancer element sequence for my gene, consisting of only about 9 nucleotides. However, when I look at pre-made constructs, they recommend multiple insertions e.g. (TGACTAA)7

How should I know how many times I should insert my enhancer, and should there be gaps in between or pure palindromic?

Tx in advance!!

[kharsh]

I am attempting to clone a gene which i have amplified thru PCR. I have added HindIII and NotI sites at its 5' and 3' ends. I have ligated this gene into a vector after double digesting both of them by same HindIII and NotI enzymes. After transforming it into DH5 alpha cells and isolating plasmid i am double digesting it with same enzymes but not getting the insert released out. I have tried sequential digestion and simultaneous digestions. But no success. However when I am doing PCR of gene using this plasmid as template then I am getting gene amplified which i can see in agarose gel. To add more to my problem whenever I am sending this plasmid for sequencing then all my reactions are getting failed! PCR amplification confirms presence of my gene but restriction digestion is not yielding me the concerned gene. Can there be a possibility of defective enzyme?? If anybody knows anything then please help me out.
Thanx

[JackBean]

you have probably some mutant, try another primers for the sequenation or to amplify your gene by primers, which anneal to plasmid and check length of your product

[JackBean]

or at least try another enzyme(s), which should cut your plasmid with insert to see, what will you get

[kharsh]

k wl try the latter option

[cassiebrazfield]

this was helpful thankyou.