Growth of E.Coli culture - please help out-i am desperate (Nov/02/2008 )

Hi All,

I am having problem with the growth of Human GABAA receptors Plasmid culture DNA. I am using LB broth and 5Ul of Tetracycline (5mg/ml) and 2Ul Ampicillin (100mg/ml) and incubating them over night. But there is no growth. I dont know what to do , If anybody can help me with it or any suggestions or have worked with them,please let me know. I will be highly grateful. I am highly tense.

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Transformation fail?
Not using E. coli CLONING host?
vector did not carry tetracycline AND ampicillin resistance gene?
Recovery period after transformation too short?
or, ligation fail?

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Yes transformation fail.
Recovery period after transformation was 1 hour.
I have been told that this strain of E.Coli carry both tetracylcine and ampicillin.

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What cells are you using? What plasmid(s) are supposedly in the cells? On which plasmid is the receptor?

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I am using E.Coli. After failure of growth we did transformation by using tetracyline and ampicilline on LB agar. The colonies did grow but when i release the plasmids using Promega KIT but , very faint (almost negligible) band were found, howevery intense bands were obtained at the top of ladder 1 KB at approximatelt 1000bp.I dont know whether these band are of plasmids but plasmids are usually 5kbs, or supercoiled or genomic DNA. can you help?

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Ok, what strain of E coli are you using? I also presume you have no details about which plasmids you're working with. It's very important to know that. Also, what is the volume of media you're using, so we can calculate the final concentration of antibiotic (you might be adding too much for the cells to survive).
Do you have an image of your gels for us to look at? Your description sounds a bit confused; do you mean the intense band is at 10 kb (the top of the 1 kb ladder)?

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You are right that i dont have enough information about all this stuff as i am new, its been two months only that i have got enrolled in PhD. But you made a very good point that i should know the strain. when my supervisor will come , i will ask her as she is currently overseas for a conference.

Yes the intense bands are at the top of the 1Kb ladder. i use 5 ml of LB broth and 5ul of ampillin (100mg/ml) and 2 ul of tetracyline (5mg/ml). I have attached the gel picture. please have a look on it and let me know.

my email address is nasiara1@hotmail.com.

i am very anxious as my time is wasting and i am unable to proceed.

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MC1061/P3 Ultracomp WAS TEH STRAIN OF E.Coli used for transformation.

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Great. What can you tell us about the plasmid? How big is the backbone? For that matter, how did you get hold of it (was it left to you by a previous student?)? If it was given to your supervisor, she should have the details from whomever sent it to her.

Your gel looks like a fairly standard miniprep. The different lanes show plasmids with differing amounts of supercoiling and nicked DNA. What is the size of the top band of the marker (it's hard to know unless you know which brand of 1 kb ladder you use in your lab).

You might want to try 50 ug/ml ampicillin final concentration, rather than 100.

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aside from reducing antibiotic concentration. How about using only one antibiotic. I assume that your plasmid gives resistance to both amp and tetracyclin.

Try transforming your plasmid, and plating the cells onto Amp only, tetracyclin only and amp+tetracyclin plate.

It could be that your plasmid doesn't actually contain the resistant gene for both amp or tetracyclin. Do you have any information on the plasmid? Size, construction history, sequence map? Since the plasmid was not built by yourself, you should have a though at the back of your mind, 'Did they send me the right thing.' It is rare, but not unheard off.

If you have enough plasmid, is it possible to validate that you do indeed have the right plasmid

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