What is your transformation method?? Did you try elecroporation?? Did you confirm that your vector and gene of interest ligation was done successfully??

Calcium chloride transformation method. Heatshock so on. I even changed the T4 DNA ligase of different companies.

For the large size, you can try electroporation, lipofection, particle balistic method, or very precisely micro injection. You also check the vector, whether your gene of interest are added or not, after your insert-vector ligation step. You can do this by simple agarose gel electrophosesis, or sequencing followed by agarose gel electrophoration and gel purification . Or you can check through restriction digestion method, followed by Agarose gel purification methods. 

Dear Sesha,

The final plasmid you are trying to construct is relatively big, and could be difficult to achieve if the ratios aren't good. Check in this web entry:

http://www.methods.info/Methods/RNA_DNA/ligation_simple.htmlHeree you'll find a hyperlink to an Excel file were you can easily calculate the amount of DNA for the ratios directly from the intensity of the bands in the gel.

Anyway you should, as Vishma suggested above, verify whether the ligation indeed occurred. One easy way would be to repeat the ligation (3 Times the volume of your regular ligation) divide it in 3 and use one volume as template for the a PCR reaction using a forward primer for the pJet vector (it comes with the kit) and a reverse primer for an internal sequence of the insert. As your cloning is not directional you might want to use a second aliquot as template for a PCR using a forward primer to an internal domain of your insert together with the reverse primer of the pJet vector. In this way you'll see amplification only when the ligation occurred. Try to get your internal primers close to the ends of the insert to reduce the PCR times. Theoretically you can use the forward and reverse primers from the pJet to amplify the insert but since it is quite big and your initial amount of template low it might not work.

Hope it helps.

Indeed the insert is quite big. Assuming the protein is non-toxic to DH5a, maybe try some "better" cells? (as I'm assuming your DH5a are homebrew :P )

better cells

https://www.thermofisher.com/nl/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/dh5a-competent-cells.html?gclid=CLrm6cKlhMoCFQw6GwodqLwILQ&s_kwcid=AL!3652!3!77674578685!p!!g!!dh5alpha&ef_id=VoGWDQAABbl3wB2k:20151230191420:s

Paul Hubert Meijboom The insert is small i.e., 214 bp. Vector/Infectious clone is big >15 Kb.

Yes, my mistake on nomenclature. Anyway your construct size is quite big right? I remember a standard pgem vector is 3k or so, so when my insert was 3.5k I more than doubled the construct size and also had difficulties (transforming 7.7k only). If i remember correctly I was lucky and got 1 colony using Mach1 competent cells (don't tell the boss, they were expensive...) 

Sebastian Dupraz The link is not working dude.  Thanks for the suggestions though.

HOME METHODS LINKS NEWS TIPS SOFTWARE FORUM CONTACT SEARCH

Not found

Sorry, the page you requested was moved or does not exist. Try again using our search.

Sorry, It got joint with the next sentence perhaps that's the problem.

Try again.

http://www.methods.info/Methods/RNA_DNA/ligation_simple.html

Inside this page check under estimation of vector and insert amounts. I use this al the time. When you have such a big vector and small inserts you might find that results from this formula gives vector: insert ratios 1:0,01. Follow the suggested results diluting your insert if necessary. 

Sebastian Dupraz Thanks for the suggestions. I will do it and let you know 

i would suggest you to increase the amount of insert  

Naaz Abbass. Dear Naaz, as per the calcuiation based on the size of the vector (>15 Kb, size of insert(214 bp), i am supposed to add 0.2ul  of insert for 4 ul of Vector ( based on the concentrations of vector n insert i got  after elution), Thats too less i thought. I tried different proportions ranging from 1: 3- 1:6. But didnt get the transformants .Finally fed up after trying for a month from December 4th, I tried incubating the ligation mixture at 4C and 16C in different ratios of 1:1, 1:2 and 1:3,  These theoretical values doesnt work sometimes. So, i added more amount of insert than the calculated values theoretically. As people say Cloning is Tricky, Based on my experience i completely endorse the statement .Indeed Cloning is Very Very Tricky. I got the ligation  at 4C more efficiently with Thermoscientific T4 DNA ligase. than with 16C. Thanks all the researchers around the world for your wonderful suggestions and infact its a proud moment for getting the clone when the success evaded  me for more than a month . 

Cool ☺ congratulations!