A Year in the Lab: Bradford Bacterial Plug Production, PFGE and PCR

Monday 16th June 2014

This week began by inoculating LB broth with single colonies from my Bradford agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. If the sample was above 0.7 abs then chloramphenicol was added to inhibit replication and the sample was left out on the bench. However, if the sample was below 0.7 abs it was returned to the incubator for another hour to allow for further growth to occur. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

IMG_5689

Tuesday 17th June 2014

Today started with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until more restriction enzyme arrives in the lab.

I then prepared and labelled LB broths ready for inoculation tomorrow morning.

Wednesday 18th June 2014

As on Monday today consisted of inoculating LB broth with single colonies from my Bradford agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

Unfortunately the QIAcube is going to be out of action for a while until an engineer from QIAgen can come to have a look at it, so I decided it would be better for me to extract DNA from my PCR reference strains by hand, through alkaline lysis as I have done before. I therefore inoculated LB broths with single colonies from my previously produced streak plates and incubated these overnight, shaking at 37ºC.

IMG_6471

Thursday 19th June 2014

As on Tuesday today started with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until tomorrow (Now my restriction enzyme has arrived!).

I then collected my overnight reference strain LB broths from incubation to begin DNA extraction. This was conducted as before, starting by ensuring I had a waterbath set at 60ºC, labelled eppendorfs and 95% ethanol on ice, along with all other prepared reagents and chemicals. I began by pipetting 1ml of incubated sample into an eppendorf, centrifuging it for 5 minutes to form a pellet at the bottom of the eppendorf. I then removed the supernatant and re suspended the pellet in 100μl of TE buffer and 2μg of Lysozyme (100μl TE buffer + 2μl 50mg/ml Lysozyme Stock). This was then incubated for 30 minutes at 37ºC with agitation. 50μl of 10% SDS was added before incubating in a waterbath at 60ºC for 30 minutes. The sample was again centrifuged for 5 minutes, before remove the supernatant to a fresh eppendorf, discarding the old. 100μl of TE buffer was added and then, working in a fume cupboard, 250μl phenol chloroform isoamylalcohol was added. Ensuring the eppendorf is closed securely it was mixed for 1 minute (The mixture will appear white and cloudy). This was centrifuged for 5 minutes, and again in the fume cupboard, the top aqueous layer was carefully removed to a fresh eppendorf. I then added 650μl ice cold 95% ethanol, and incubated on ice overnight.

IMG_4989 Saturday 21st June 2014

I started today by inoculating LB broth with single colonies from my Bradford agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

I then finished off my DNA extraction from yesterday, by centrifuging for 5 minutes. I removed the supernatant and re suspended the pellet in 70% ethanol. Again I centrifuged for 5 minutes. The supernatent was then removed and discarded, before air drying the eppendorf until all ethanol had evaporated off. Finally 30μl of distilled water was added and allowed to incubate at room temperature for 5 minutes.

In preparation for running my first batch of new plugs tomorrow I cut each the first 14 plugs from the Lincoln collection in half, placed them into a fresh eppendorf and incubated in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

Sunday 22nd June 2014

As before, today started with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until they can be restriction digested tomorrow.

I then collected yesterday’s bacterial plugs from incubation, removing the buffer/enzyme before incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.

IMG_5085

This gel was then loaded with the plugs, PFG and Lambda ladders before running overnight using the following settings:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 40-100 switch intervals per second
  • 6 V/cm
  • 30 minute stain, 10 minute destain

I ran this gel using the same settings as my last most successful PFGE gel as confirmed last week.

In preparation for running my next batch of new plugs tomorrow I cut each plug in half, placed it into a fresh eppendorf and incubated them in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

Based on my failed first attempt at PCR last week I decided to try again with my new positive control sample. Again I just used my CTX-M group 1 primers, however I changed my positive control to the reference strain DNA from NCTC 13353, using ATCC 25922 DNA for a negative control. I carefully pipetted my required reagents into 50μl PCR tubes, including mastermix Taq2x, primers, plasmid DNA and pure water. I then placed this into the PCR machine and conducted a temperature gradient, ranging from 49.6ºC to 62ºC in order to optimise the most suitable temperature for group 1 forward and reverse primers.

IMG_6703

After 2 hours my PCR finished and I ran the resulting PCR product on a 1.8% agarose gel for 1.5 hours, alongside a 50 bp ladder for reference. I stained the gel for 15 minutes before viewing under UV. The resulting gel was much better than previously, demonstrating clear and accurate banding for both my 50bp ladder and samples. My negative control showed no banding, and my temperature gradient demonstrated that 62ºC was the most appropriate temperature out of those tested. I did notice however that my banding was very thick and dark. This could be as a result of using too concentrated primer and/or DNA.

PCR CTX-M Group 1 NCTC 13353 22.06.14

Next week…

I will be continuing to produce bacterial plugs from my Bradford samples, conducting restriction digests and then running these plugs, along with my previously produced Lincoln plugs, through PFGE to analyse strain. Based on my PCR work this week I plan on conducting both primer and DNA concentration gradients, as well as using the Nanodrop to analyse the purity and amount of DNA present within my extracted NCTC 13353 samples.

A Year in the Lab…

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