donny

I am planning on inserting some genes into the chromosome of E. coli. How should I choose where to insert the genes? What are the easiest methods for gene insertion/replacement in E. coli? Some references will be nice. Thanks!

I was moving to a new lab and brought my bacteria strains over by air drying cultures of them on filter paper. I wrapped them up in weighing paper and aluminium foil on the outside. I carried it in my hand luggage (ie went through all the X-ray). It was not exposed to extreme heat. For most of the trip, it was ~25C and when I got to my destination, it was mostly below 15C. However, when I finally got to the lab after ~12 days, I could not revive them. What I did was I added a drop of LB to the filter paper, incubated at 37C for 1 h and smeared it on selective plates. Then, I dropped the paper into selective liquid media. There was no colony or cell growth at all.

I know that inoculating bacteria on filter paper for mailing is a common practice. There is this thread from this forum (http://www.protocol-...posts/7295.html). Also, CGSC sends out KEIO collection strains on paper too. So what did I do wrongly? Should I have sent them moist or recover in non-selective medium first? I'm trying to figure this out so I could get people from my lab to send some over to me. Thanks!

I need to clone a very small DNA fragment encoding a small peptide (~35bp) into a vector with a fusion protein tag. I'm thinking since this is such a small fragment, I can design two oligos such that they will anneal and the sticky ends will be as desired. I can then clone the hybridised DNA into the vector. That way I can do away with digestion with restriction enzymes and avoid difficulties in purification since the lowest cutoff for spin columns is 40bp. Does this make sense? Has anyone tried this?

I've been screening mutant libraries and the libraries keep ending up with the same mutant. I made new libraries and the same thing happened again. I suspect 2 sources : reused electroporation cuvette and aerosol during pipetting. I can easily use new cuvettes but I seriously doubt it is the main cause because I've tried transforming cells in the old cuvettes just to test the sterility and got no colony. Moreover, I exposed the cuvettes to UV at 300nm over the illuminator for DNA gel for 10min before using them. I guess that should damage whatever residual DNA that could have transformed. I'm thinking if aerosol generated by pipetting during cell preparation could have caused the cross-contamination. However, instinctively, I find it hard to believe the (invisible) aerosol could cause such extensive contamination. Has anyone experienced cross-contamination due to aerosol generated by pipetting?

I am trying to generate a mutant library for a 7.4kb plasmid from ligation. I've been working with DH5a and recently got hold of some XL10-Gold, which supposedly gives better transformation efficiency for large plasmids and ligation products.

1) XL10-Gold is sold as a chemical competent cell for large plasmids and ligation products. Can I prepare electrocompetent cells from the stock? Would it perform as well as the chemical competent ones? Just curious why it's not sold in the electrocompetent form as well.

2) If I prepare electrocompetent DH5a and XL10-Gold with the same transformation efficiency using pUC18, would XL10 transform better for larger plasmids?

Thanks!