I work as a lab tech prepping university classes; I don't get a lot of opportunity to get some microbiology experience, and I thought I would take advantage of the extra down time we have since the semester was ended early.
I took on a little project of seeing what kind of bacteria are growing on my pet chinchilla. I do not know what the bacteria is.
I have 5 samples; 4 are gram-positive coccus bacteria and one is a gram-negative rod bacteria after doing a gram stain. The 4 coccus bacteria are circular colonies, ranging from white-ivory-orange. The rod bacteria appears to be a rhizoid colony that is semi-transparent (these were all grown on YPD agar plates).
I just followed the Qiaprep spin mini prep protocol where I used 2mL of bacteria to extract DNA. Because I am unsure what exactly the bacteria is, I decided to do the step where you wash the spin column with 500uL of Buffer PB.
What should my agarose gel and DNA sample that I load look like? I want to see if the extraction was successful.
This is my second attempt at DNA extraction for this project; the first time I ran a 2% agarose gel with sybrsafe at 180V for 40 minutes, using 6uL of a 1KB ladder and 1uL 6X LD/5uL DNA sample (extracted from 1mL of culture) and only got a single, extremely faint band. I thought maybe the sample was too little, so I tried it again at 3uL 6x LD/15uL DNA sample but got the same result.
I've been able to understand and kind of make my own protocol up till now; I'm proud of myself for doing a successful gram staining, considering it was my first time ever trying it (btw, neutral red seems to be an alternative counterstain if you have no safranin or fuchsine available). I just don't have a lot of experience working out these kinds of problems (hence why I'm trying this). Is there something I'm doing wrong here and how should I proceed to get a successful DNA extraction?
About 10ng of dna in a single band is visible with ethidium bromide dye. Smaller amounts are possible with small combs,single band and short runs and better dyes . The problem with genomic dna is that either it is intacct so very large and never leaves the well of the gel just giving a bright stain on the anode side of the well having gone nowhere or that it is degraded by enzymatic and mechanical shearing so is randomly spread over a wide size range and no individual part of the smear is visible unless you load large amounts of degraded dna. The presence of dna is usually measured on a spectrometer at 260nm and its purity from protein contamination given by the ration of OD260/280 which should be about 1.8 for pure dna
Paul Rutland I do have a nanodrop at my disposal! Since the last step of the protocol was to add buffer EB to elude the plasmid DNA from the column, would I use plain buffer EB as my blank?
Kitt Kroeger nanodrop is very sensitive to any difference between samples and it is essential always to blank/zero nanodrop with exactly the same solution that the dna sample is dissolved in. In your case this is EB. Blanking with any other solvent results in wrong readings and can often even show dna concentrations less than zero so yes...use EB
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