Hi everybody, can anybody tell the maximum size of DNA, which can be inserted in pTZ57R and pFL61? I have a 3.1 Kb fragment amplified from genomic DNA, I want to clone that..
Hi Prabuddha
Most of the plasmid vectors should easily take the fragment of your interest...3.1 kb
pTZ57R is ~2.9 kb and should easily accommodate inserts double its size. Anything more should be a bit tricky but not impossible.
Should be no problem with 3.1 kb, but I wouldn't try anything much longer
Hi prabuddha,
The 3.1Kbp fragment is fine to be inserted in any of the plasmids you have mentioned. Normally such plasmids can take an insert size of upto 10Kbp. So you are well under the limits.
The uptake of insert by plasmid is not dependent on size, as far as the insert is less than 10Kbp. But what you have to remember is that while cloning make sure that you purify you product before cloning, just to make sure you are not cloning any unwanted smaller fragments from PCR, which tend to have higher efficiency that large insert.
Hope it helps
The amplified genomic DNA fragment of 3.1kb could be cloned comfortably into both vectors. If you encounter problems during cloning, then you could check other parameters, like your vector/insert purification, vector dephosphorylation and ligation reactions, insert to vector ratio, e.t.c. In the absence of E. coli transformants, you could also try other E. coli host for transformation reactions, like TOP10, HB101. I guess these suggestions would be of assistance to you.
You can insert into both vector between 3.1 to 3.8 kb depends upon different paramters.
There is no problem,,, but it is better if you increase the amount of bacteria after ligation,,, it would be better and easier!!! : )
You can easily ligate the 3.1 kb insert in your vectors. I think there will be no problem if you make sure that all the required parameters are taken care off.
Both the vectors you can use for cloning. The problem with the huge insert is, you will get very few colonies
You can reduce empty vectors by optimizing the ratio of vector DNA to insert in the ligation
reaction, dephosphorylating the vector, or using directional cloning.
I am using TA cloning. That works well. Thanks for all the inputs again.
It shouldn´t be a problem the size of 3.1 kb, just be sure to maintain the stoichoimetry of insert and vector properly 3:1.
According to Fermentas, which sells this vector, the maximum size is 10 kb. They sell the InsTAclone PCR Cloning Kit (K1213 e K1214) including the vector, so I think using the kit you can clone up to 10 kb. They do not sell pFL61, but they think it is the same maximum size. Good luck for your jobs!
Sorry but I do not have experience about it is possible to insert your DNA in pTZ57R and pFL61. Good luck in your job.
Thanks. I have the kit. I use that only. pFL61 I got from other sources.
Its not the size what matters but see the ratio of the vector and insert if maintained size should not be a barrier
I have problems with the clonation of a fragment of 1.3 Kb cloning with pGEM Teasy and clonJET.
I couldn't get enough concentration of my insert, so couldn't manage to keep the ratio as 3:1. May be it is 1:1. Ligated at 4C overnight as fermentus recommended. Then electroporated. After 12 hrs, I can't see anything. May be have to wait for another 5-6 hrs....hope for the best.
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