• Make sure you use your insert to vector at a ratio of at least 3:1
• make sure that you pick and miniprep at least 6 colonies from your transformed LB agar plate: it is not uncommon to find that just 1-2 are successful recombinants
• Some times your method of cloning can have an effect: generally sticky ends created by restriction digest are ligated more efficiently than blunt ended products
• Finally ligating for longer periods at lower temp - even though modern formulations tend to suggest short ligation incubations - can sometimes improve ligation efficiency:
• Soecifically ligation in the fridge over night or at 16C overnight as was traditionally carried out can still sometimes yield more colonies and a higher proportion of such colonies that incorporate the insert

Ahh, i will these brilliant suggestions.

If you don't mind, just as an additional question, as I am actually a geochemist, do you mean an "insert to vector ratio" is the dna to vecotr i.e. if i use 5ul DNA as oppose to 3ul I need to increase the vector amount used?

Thank you!!

Dear Kirk

As a geochemist you are far more used to the exactitude of quantitative science and that what my answer refers to

In essence I mean an approximation to 3 parts or molar equivalents insert 1 part vector. In the old days when I did this routinely (the 80s) it meant examining the intensity of the purified cut that is linear vector in an agarose gel (sybr stained or back then ethidium bromide stained) and also a small fraction of the purified insert to ligate and making sure the latter was at least 3 x as bright as the former. Crude but effective. After all experimental biology is not physics so these sort if approximations are sufficiently accurate for the experiment to work

I should also add that cloning doesn't always work so I have been known to repeat 2 to 3 times in order to obtain a successful recombinant clone

I completely agree with Laurence beautiful thump-rule explanations given for cloning. I have an additional point to mention. Majority cloning vectors have a blue white screening strategy to identify the colonies that have insert from the plate itself. As per my understanding you used a pGEM®-T Easy Vector Systems for cloning your product. The pGEM®-T Easy pre-linearized Vector contains 3´-T overhangs and I trust you used normal Taq polymerase (Not Taq with proof reading activity) to PCR amplify your gene. If you use the later enzyme (Taq with proof reading activity), it will not create an A' over hang in your PCR product and in your ligation reaction it will not get ligate and you will not get any colonies.

My suggestion would be -

Please make sure you are using the right enzyme for your PCR reactions.

As Laurence said the insert to vector ratio of at least 3:1 or even 5:1 you can try.

You can add X-Gal (concentration - 40mg/ml) to your agar plates and spread it and dry it before plating your transformed culture. This will give you a direct additional selection and you can reduce your PCR work load.

Last not the least, as Laurence said cloning doesn't always work first time even after following all these thump-rules. If you ask why, I hope no one can give a specific answer. You need to repeat couple of times.

Good luck.