Procyon

Hello,

After I obtained the results from sequencing, I proceeded to curate the sequences and identifying (with Vector NTI) the primers I used in the sequence. The primers, although are degenerated, were found with no mismatches.

The problem is that when I blast the squence using BLASTn or BLASTX, the results are malate synthase, a gene/protein that I totally didn't expected and totally unrelated to the gene I'm interesed. These results were like 13 of the 20 sequences while 5 were of the gene I was expecting.

Please tell me what went wrong. I'm a bit desperate. I used a clone library with pGEM and chemocompetent DH5-alpha.

I'm trying to implement a H2S (hydrogen sulfide) measurement for thermal water in an in situ sample collection by an environmental microbiology and microbial ecology laboratory. I need to stabilize the H2S in the water sample to measure the H2S levels later.

Due to the higly volatility of H2S, I'm evaluating (and understand the theory of) the methods of blue methylene or diamine; but I don't know the practical limitations for an in situ sampling neither what is the proper container for water with volatile H2S.
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Hello,

I'm trying to clone a PCR product of 750 bp amplified from DNA extracted from environmental samples. I used a "Wizard PCR and Gel clean-up system" to purify the gel band of my product (I'm always careful to expose the gel to UV less time as possible). For transformation i'm using chemocompetent DH5-alpha, with a heat-shock of 42°C at 45 seconds.

I'm using pGEM or pJET as vector, but I get less than 40 transformant colonies (but I need 100+ for clone library). In my transformation control I get always 100+ colonies ( I use 1 ul of  miniprep purified pGEM with an insert) ; but when I use a ligation control for pJET, I also get 40 or so transformants.

I've previously done the ligation and transformation using pJET and DH5a with a fragment of 370 bp also amplified from environmental DNA; obtaining 647 transformants with my insert.

I suspect there is a problem with ligation, based on the controls. Is there an upper limit of insert DNA (purified or direct from PCR) quantity during the ligation? Should I increase the time of ligation?

I've repeated the PCR + ligation + transformation changing different parameters, but I still get less than 40 (white when i use pGem) colonies. I really need some advice of what to do or what may I have forgotten.