I want to clone miRNA (pre-miRNA) of 250bp. Firstly I cloned in pJET 1.2 vector with NcoI and BglII restriction site added in pre-miRNA sequence. Cloning in pJET1.2 vector is confirmed by sequencing and digestion. further I want to subclone it in pCAMBIA 1302 vector but I am not getting any positive colonies. For cloning in pCAMBIA I performed sequential digestion of plasmid obtained from pJET (for 50 ul of rxn.:10 units of enzyme (~1ul enzyme each), 1ug of plasmid, 5ul buffer (3.1 buffer) is used and incubated at 37°C). I am getting perfect dropout but further pCAMBIA transformation is failed ( for transformation, ligation is done with 50ng of vector in 1:3 vector:insert ratio at 16°C for overnight then transformed in DH5 alpha competent cells). Please suggest me how it can be done?
Personally I don't like purifying small DNA fragments after digesting a plasmid because the yield of agarose purifications are usually quite poor. Instead I always use ELIC method (Article Homologous Recombinatorial Cloning Without the Creation of S...
)I think it will work for you as you had no problem amplifying your premiRNA to clone it in pJET1.2. I normally use DH5a and my homologous fragments are about 15-18bp. The method is quite simple and always give good results.
If you amplify your PCR fragment using as template pJET1.2 with your premiRNA you don't even have to load a gel to purify your amplification, you can directly use a column to purify your PCR product and use that as insert to mix with your digested pCAMBIA vector. In the paper they stated that you need to get rid of your template after PCR, but as pJET is AmpR and pCambia is KmR, colonies transformed with pJET will not grow after your transformation.
Good luck!
If a large number of negative colonies appear, it is likely that the restriction enzyme treatment of pCAMBIA1302 is insufficient, resulting in self-ligation.
If colonies do not appear at all, then the restriction enzyme treatment of the insert is probably insufficient.
With which restriction enzyme are you treating pCAMBIA1302?
How are you adjusting the miRNAs from pJET1.2, is it PCR? Is it restriction enzyme treatment? Also, how do you purify the 250 bp fragment?
If you are using PCR, do you have extra sequences at the end of the primers other than restriction enzyme sites?
Best,
Narumi
- Badges
- Science topic