Why don't I get the expected entry clones when I use the gateway system?? - (Apr/28/2012 )

Hi to everyone,

I am a new user and I would like to ask for some help on the gateway cloning system from anyone who has successfully used it before, have some experience on it and would like to help. My question is very specific...
I am trying to insert my AttB PCR product into pdonr221 in order to make the entry clones. For the first three genes it wasn’t that difficult. However, I still have three more genes to clone and I am stuck on this for three months. After the BP reaction I used to check by digestion 10-20 colonies but now I have reached 50-70 and I still can get the right digestion pattern. I have sent some for sequencing and I found out that between the att regions I get a piece of my gene or a small portion from the start and a small region from the end. I can understand why most of the clones are lacking a part from the middle of my gene sequence. I have been through the whole invitrogen troubleshooting but I haven't been able to solve my problem. I would really appreciate any suggestions that you can make.

I think that I should give some more details as well...
1) the inserts are less than 4Kb
2) they are gel purified
3) I have already checked a portion of the PCR products by gel electrophoresis and sequencing in case that might be some breaks from the UV during the band excision
4) vectors and competent cells also checked

I can't think of anything else. If anyone had the same problem in the past and have solved it please let me know.

Thanks

You might check the sequence of the genes you are trying to insert to see if they have att -like sequences.

Hi,
thanks for your advice but I have already checked this as well and it is not the case. Any other suggestions?