AquaPreserve reagent (www.aquaplasmid.com/AquaPreserve.html) works very well for RNA extraction from frozen EDTA, ACD, heparin, and other anticogulated whole blood samples.
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700 ng/ul is a lot of DNA for a BAC miniprep! How much culture did you use for the miniprep? How much water did you use to solubilize the DNA? Do you mean that you saw DNA in the well but not in the gel lane for the uncut? Did you see any DNA in the well after EcoRI cut? If not, you might have gDNA contamination but didn't have the BAC.
We found that more than half of the denatured plasmid DNA could not find and re-anneal with its partner strand after being denatured during alkaline lysis. So we developed a potent non-alkaline lysis method for plasmid extraction and turned out it improved pDNA yield many folds. Your megaplasmid, same for BAC, might be even harder to re-nature after alkaline lysis and they may get lost like genomic DNA in the cell debris after neutralization. I think our non-alkaline lysis based AquaPlasmid could help. If you are interested at trying it out, feel free to contact us.
Tihong, do you have any luck in getting the plasmid from Pseudomonas aeruginosa? What method or kit did you use to do the preps? Could you show us your gel? How intense was the smear? I assume that you had RNase A in the cell suspension buffer and the smear wasn't RNA. Maybe the plasmid wasn't replicate well in PA, or maybe PA has strong nucleases, or maybe your alkaline lysis was't potent enough to lyse the PA? If boiling won't help, I would like to suggest AquaPlasmid, which now uses a novel non-alkaline lysis mechanism to prevent pDNA denaturation during cell lysis and increase pDNA yield.
Interesting project! But some phages might be carried into blood via macrophages that eat up bacteria infected with your phages in the gut.
Washing/cleaning the larva off phages is nearly impossible. There are millions of phages on the surface.
Fish on slides can show the distribution of phages but you couldn't recover the phages that have entered the blood stream for sequencing.
You couldn't use antibody binding to select those entering the blood as all of them have your inserted coat.
Just curious. Are you trying to identify blood-entering phages for treating bacterial infection?! We have used directed evolution to generate phages that can overcome phage-resistant bacteria by random mutagenesis by treating the resistant phages with AquaMutant solution. Maybe you could also create mutant phage library using AquaMutant to increase your odds of finding phages that can enter the blood.
Hope you find a way to separate phages in the blood.
I assume you have the gene cloned in an expression vector and have a functional assay established to access its activity. You could use site-directed mutagenesis but it may be too much work and too many sites to test. And worst of all, you may end up not finding the most critical or all of the critical residues of the protein, if any. Random mutagenesis would allow you to create a mutant library blindly and screen for the mutants that show varied activity. Subsequently you would sequence those that show interesting activities, be it loss or gain of function, to see which residues might be critical to the function of the protein.
For random mutagenesis, you could use error-prone PCR-based method to create the mutant library, which involves the use of mutagenic PCR to amplify the gene, cut and paste back into the vector for expression. However, if the gene is large this method is very inefficient and the mutant library is often not fully representative.
We have a new random mutagenesis reagent kit and method, AquaMutant, which is chemical-based. You may either soak your cut out gene with AquaMutant to introduce random mutations or you may treat the amplified PCR product with AquaMutant to introduce random mutations. The mutation rate can be varied and controlled by the concentration of AquaMutant (from 1-100%) and the exposure time (from 1-30 min). Subsequently you would clone the mutated gene back into the expression vector.
If you hate the cutting/pasting cloning workload, which can be time-consuming and frustrating, you could try simply soak the isolated plasmid DNA directly in AquaMutant to get the mutant library. Of course, you would have mutants with mutations in the plasmid other then the gene of interest and you would lose mutants with mutations in the antibiotic resistance gene or origin of replication, but you can still get a library that is more than large enough and fully represented due to the use of micrograms of plasmid DNA for the random mutagenesis.
In addition to the above in vitro random mutagenesis approaches, you could use AquaMutant to treat the cells harboring your episomal expression vector in an in vivo random mutagenesis. You would then use FACS sorting to pick out and separate those cells expressing your protein for activity assays. You don't need to care about those junk mutations elsewhere in the cells for the moment. After you have sorted out the interesting clones, you could re-introduce the episomal mutants into the original cell line to confirm their activities. The options are all yours.
Best of luck!