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Gateway cloning


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#1 Rits

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Posted 25 August 2009 - 06:42 AM

Hi there

anyone expert in Gateway cloning there??

need some help as I am trying to make some changes in an exisiting destination vector ( created by some other person), and with ccdB box and CmR gene there.

I am finding it impossible to transform this existing destinatuion vector as it should grow on the Invitrogen supplied ccdB survival competent cells, as they have discontinued the DB3.1 cells now :-(

Can anyone please help as its really frustrating, is there any other available competent cell line to transform these vectors with ccdB suicide box????????

please help :lol:

#2 mcast42

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Posted 25 August 2009 - 09:18 AM

Which antibiotic are you using? The Gateway cassette has Chloramphenicol resistance but it might be better to use another selection agent. I too am having a difficult time making a destination vector. I have had somewhat better results after switching to amp plates instead of chloramphenicol plates. I also found that letting them recover a bit longer than 60 minutes and growing longer on the plates than DH5a or Top10s (more like 20 hours instead of 16). My controls now grow - still working on the experimental.

Good luck! I can only hope that making the final expression vectors is worth this trouble.

#3 dpo

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Posted 26 August 2009 - 05:58 AM

Invitrogen replaced the DB3.1 cells by One Shot® ccdB Survival™-T1R Chemically Competent Cells (Cat. No. C7510-03) that can be used to grow Gateway destination vectors.

good luck

#4 Rits

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Posted 26 August 2009 - 08:14 AM

Invitrogen replaced the DB3.1 cells by One Shot® ccdB Survival™-T1R Chemically Competent Cells (Cat. No. C7510-03) that can be used to grow Gateway destination vectors.

good luck


Yes , I got those and tried these new cells from Invitrogen, but my transformation plates are like a couple of colonies, I am following the protocol, long incubations, everything as comes on the manual with these calls but the transformation efficiency is pathetic, theres no way this system would work when I shall need to make some SDM on these vectors!!! Don't know what to do, its very frustrating. I have used chloramphenicol as antibiotic, also did amp and kan as parallel for transformation, nothing is working any better!!!!

Does anyone know about any other competent cell line from some other company for teh survival of ccdB containing vectors!!!!

Don't know where I am going wrong :o

#5 Rits

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Posted 26 August 2009 - 08:16 AM

Which antibiotic are you using? The Gateway cassette has Chloramphenicol resistance but it might be better to use another selection agent. I too am having a difficult time making a destination vector. I have had somewhat better results after switching to amp plates instead of chloramphenicol plates. I also found that letting them recover a bit longer than 60 minutes and growing longer on the plates than DH5a or Top10s (more like 20 hours instead of 16). My controls now grow - still working on the experimental.

Good luck! I can only hope that making the final expression vectors is worth this trouble.



Used chloramphenicol as well as the ampicillin and kanamycin plates, nothing is any better than other :-(

I have used 60 minutes recovery period so far shall try longer this time if that will make it any better

Rits

#6 albatross

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Posted 27 August 2009 - 07:50 AM

I also fail to make a detination vector by using the Invitrogen ccdB survival competent cell and Amp plates.
And have not found any method overcome it.
what is the "recovery period"?

Which antibiotic are you using? The Gateway cassette has Chloramphenicol resistance but it might be better to use another selection agent. I too am having a difficult time making a destination vector. I have had somewhat better results after switching to amp plates instead of chloramphenicol plates. I also found that letting them recover a bit longer than 60 minutes and growing longer on the plates than DH5a or Top10s (more like 20 hours instead of 16). My controls now grow - still working on the experimental.

Good luck! I can only hope that making the final expression vectors is worth this trouble.



Used chloramphenicol as well as the ampicillin and kanamycin plates, nothing is any better than other :-(

I have used 60 minutes recovery period so far shall try longer this time if that will make it any better

Rits



#7 kuddus987

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Posted 28 August 2009 - 03:02 AM

The Gateway cassette has Chloramphenicol resistance but it might be better to use another selection agent. I too am having a difficult time making a destination vector. I have had somewhat better results after switching to amp plates instead of chloramphenicol plates. I also found that letting them recover a bit longer than 60 minutes and growing longer on the plates than DH5a or Top10s (more like 20 hours instead of 16). My controls now grow - still working on the experimental.

Shamwow

#8 kuddus987

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Posted 28 August 2009 - 03:05 AM

I got those and tried these new cells from Invitrogen, but my transformation plates are like a couple of colonies, I am following the protocol, long incubations, everything as comes on the manual with these calls but the transformation efficiency is pathetic, theres no way this system would work when I shall need to make some SDM on these vectors!!! Don't know what to do, its very frustrating. I have used chloramphenicol as antibiotic, also did amp and kan as parallel for transformation, nothing is working any better!!!!

Resveratrol

#9 kuddus987

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Posted 28 August 2009 - 03:08 AM

I have had somewhat better results after switching to amp plates instead of chloramphenicol plates. I also found that letting them recover a bit longer than 60 minutes and growing longer on the plates than DH5a or Top10s (more like 20 hours instead of 16). My controls now grow - still working on the experimental.

Colon-cleanse




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