I've been growing TG1 bacteria in M9 minimum salt plates for 36 hrs. picking a colony and grow it in 2xTY media overnight. Next day I'm starting a 100-fold diluted culture and growing it until it reaches 0.5 OD. After that, am infecting with helper phage dilutions to do a helper phage titration. Incubating the bacteria 30 min at 37 C in a wather bath and then plating the bacteria in TYE Kanamycin plates (50 mg/ml), but everytime I hvae done it I get bacterial grow in my negative (No helper phage).
I have tested the TG1 multiple times and still getting the same results. Any thoughts or recommendations?
There could be a contamination of your TG1 contamination with either helper phage or with cells transformed with any kan resistance plasmid used in your lab. Could you try another source of TG1 cells? If this is the case, the problem can be easily fixed by changing the bacterial stock.
If the contamination is not limited to the original cells, but every cell you grow gets contaminated, the problem is a bit more tricky, but can be solved. Are you using filter tips? I would strongly recommend to do that when handling phages. Are you decontaminating glassware and plasticware with bleach? That can keep contamination under control. I soap everything in 2% bleach overnight. Besides that, you would have to discard any biological materialor chemical that can be already contaminated. Just being careful and following such simple rules, you can eliminate contaminations in the future.
Gertrudis have you used Daniel christ Dab library before? Would you have any recommendations on libraries?
Gertrudis Rojas Have you used Daniel Christ Dab library or know someone has successfully use it?
I havent used Daniels's library. Did you have any problem?when using it?
Edwin Velazquez Hi! we use Christ Dab library in my lab, you can write to me for further info!
Eduardo García-Calvo How was your experience? I had to do lots of troubleshooting at the beginning it was kind of a hell. I finally made it work like a year ago, but only for phage ELISA and I have been isolating positive clones since then. However, I was never able to successfully produce the soluble fragments with HB2151 cells. so I frequently get ambar stop codons in the CDRs. I have successfully sequenced fragments but, only If I grow colonies from the positive clones that have not been infected with helper phage yet, otherwise is a mess. So from the original overnights I do streaks and then if positive again from the overnight culture I miniprep and sequence and clone into pET-scFv-T from addgene. The fragments are easily purified with His-tag or protein A although Protein A gives less yielding compared to His-tag. How has been your experience in general?
Gertrudis Rojas It was rough at the beginning. It took me like a year to troubleshoot the whole thing. It only has been working for me with phage ELISA, but not for soluble yet. Sometimes I get signal in the blanks if I do not block overnight or at least 45 min RT and 15 at 37 C. I do not know if they send me the right HB2151 strain, but everything else I have made it work. I frequently get ambar stop codons though, but I have overcame the problem by cloning the fragments into rosetta blue cells. What library do you use? I tried to get Tomlinson library too, but no one distribute it anymore. Can you share yours with an MTA? I would love to test another library, but they are very pricy like 25 K dollars and we do not have that much funding at this moment.
Getting plenty of amber codons is a common finding when using synthetic libraries. They form part of the library because of the use of NNK and NNS triplets and seem to have a clear advantage during phage selection. It happens with most synthetic libraries and does not preclude their use . I always replace TAG before going to soluble expression, in my case in mammalian cells.
It would be very easy to make your own synthetic library. See the detailed protocols by Sachdev Sidhu. Making a naive library is more difficult but not impossible. . If you use trinucleotide synthesis for the synthetic library, stop codons will be absent, but synthesis is expensive.
Edwin Velazquez we have "suffered" all the troubles you mentioned. I have learned that we have to be very careful with the phage display results because is not easy to "translate" them to soluble (even the "good ones"). And yes, the are a lot of false positives and reaction with blocking buffer (we have use casein instead of BSA for solving it). We have done the cloning into pET-ScFv-T and the producing it in BL21 pLys with good yields. We are building now our own libraries (immune in my case) and it seem that may work better, we searched for a lot of commercial libraries, like tomlinson i+j with good results but i don`t know if it is still available. We also were thinking to buy ALTHEA which cost like 6k for research purpose. Hope this helps you :)
I was involved in the review of the ALTHEA library manuscript and I found the library OK but not great and probably not worth 6 K. Synthetic antibody libraries might look easy to generate but you ARE going to have a lot of problems with the developability of the selected hits unless it's just for academic purposes and positive hits in phage ELISA is your final "product". Making antibody libraries, good ones are NOT trivial at all, and that's why high-quality ones go for millions of dollars, not a couple of thousands.
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