Valdría la pena probar?

Pimp Your Plasmid Growth Medium

April 28, 2008 | By Nick In Tech Tips |

I often wonder why it is that molecular biology researchers stubbornly refuse to change 40 year old methods that, while they work, are not as good as newer, faster and cheaper methods out there.

I suppose rational scientists have often irrational superstitions.

One example of an old method that could be improved is the growth media used for plasmid preparation.

The majority of us, throughout our university careers, have used either SOC, LB or TB, for recombinant plasmid propagation, typically in E. coli. LB or Luria-Bertani broth has been in use for almost 60 years or thereabouts, while SOC has certainly been in use for 2 decades.

But by adding in a few more ingredients or being more economical on others (especially yeast extract and tryptone) that you could get a higher plasmid yield, quicker and with less money.

To counter the naysayers, nobody wants to make very complex with 15 ingredients requiring filter sterilisation, as this obviously defeats the object of economy of time and budget. Indeed, there are trade-offs between optimising for biomass, plasmid yield, quality, stability and cost with the difference between protein production and plasmid production being that plasmid production requires only cell growth, division, and plasmid stability.

The good news is that Michael Danquah and Gareth Forde from Monash University down-under have devised a stoichiometrically optimised medium for plasmid production. PDM, supposedly yields under the conditions they tested, twice the amount of plasmid in both volumetric and specific yields compared to TB , LB is left in the dust. Better yet, because it uses less tryptone and yeast extract, the cost per mg of DNA is roughly one quarter compared to LB.

The recipes for LB, TB, SOC and PDM are shown below. If you decide to break with tradition and give PDM a go, be sure to tell us how it goes.

Note – Autoclave glucose, KH₂PO₄ and Na₂HPO₄ separately

Peligros de la contaminacion con micoplasma

Oncogene advance online publication 14 April 2008; doi: 10.1038/onc.2008.103

Mycoplasma infection suppresses p53, activates NF-B and cooperates with oncogenic Ras in rodent fibroblast transformation

Prokaryotes of the genus Mycoplasma are the smallest cellular organisms that persist as obligate extracellular parasites. Although mycoplasma infection is known to be associated with chromosomal instability and can promote malignant transformation, the mechanisms underlying these phenomena remain unknown. Since persistence of many cellular parasites requires suppression of apoptosis in host cells, we tested the effect of mycoplasma infection on the activity of the p53 and nuclear factor (NF)-B pathways, major mechanisms controlling programmed cell death. To monitor the activity of p53 and NF-B in mycoplasma-infected cells, we used a panel of reporter cell lines expressing the bacterial -galactosidase gene under the control of p53- or NF-B-responsive promoters. Cells incubated with media conditioned with different species of mycoplasma showed constitutive activation of NF-B and reduced activation of p53, common characteristics of the majority of human tumor cells, with M. arginini having the strongest effect among the species tested. Moreover, mycoplasma infection reduced the expression level and inducibility of an endogenous p53-responsive gene, p21^waf1, and inhibited apoptosis induced by genotoxic stress. Infection with M. arginini made rat and mouse embryo fibroblasts susceptible to transformation with oncogenic H-Ras, whereas mycoplasma-free cells underwent irreversible p53-dependent growth arrest. Mycoplasma infection was as effective as shRNA-mediated knockdown of p53 expression in making rodent fibroblasts permissive to Ras-induced transformation. These observations indicate that mycoplasma infection plays the role of a p53-suppressing oncogene that cooperates with Ras in cell transformation and suggest that the carcinogenic and mutagenic effects of mycoplasma might be due to inhibition of p53 tumor suppressor function by this common human parasite.

tips para minipreps

http://bitesizebio.com/2008/04/08/5-more-tips-for-dna-gel-extraction/

Quick and dirty

Identificacion de plasmidos con inserto en clonaciones

Reagents:

2x Lysis Buffer:

20% w/v Sucrose
200mM NaOH
120mM KCl
10mM EDTA
0.5% SDS
a pinch of Bromophenol blue
store at -20 degC

Procedure:

There are two ways of performing this screen. If the chances of you having a clones depend on the number of colonies you screen i.e. your “luckiness” factor is abysmal, then obviously you would have a screen a large number of colonies. Then go ahead with the 96 well plate method. If you are the type who usually strikes on the clone in a screen of say 10 to 20 colonies then you could use Eppendorf tubes.

1. Dilute lysis buffer to 1x, at room temperature.
2. Pick the colonies from the transformant plate “patch” to a master plate and transfer remaining to 30 microL of lysis buffer (in Eppendorfs or a 96 well plate)
3. Incubate at 37 degC for 5 to 7 minutes
4. Chill on ice for 5min
5. Spin at maximum speed for 10 minutes ( you would need a compatible centrifuge to spin 96 well plates - which should be sealed with parafilm)
6. Load 10 to 15 microL on the gel (Caution: When trying to load from the lysate make sure you only pick up the supernatant. The gooey mass below (caused by genomic DNA) will stick to your piptte causing pipetting difficulties as well cause the sample to jump out of the agarose lanes. If this happens spin down again for a few minutes and load)
7. Difference in electrophoretic mobility will help you narrow down your clones for mini preps.

De
http://bitesizebio.com/2008/04/02/quick-and-dirty-screening-for-cloned-inserts/