Monday 31st March 2014
This week began with the inoculation of LB broth with single colonies from my Lincoln Escherichia coli nutrient agar slope collection (E1 – 48). These broths were then shaken and incubated overnight at 37ºC. I then decided to make more LB broth as before by preparing, decanting and autoclaving the broth in glass universals.
The excessive blurring seen on my last PFGE gel indicated that the plugs contained too much DNA, that hadn’t been properly lysed and therefore were also not appropriately cut during incubation with restriction enzyme. In order to ensure that this new batch of bacterial plugs do not contain too much DNA I decided to grow Escherichia coli and measure the optical density (OD) over time to achieve a lower concentration of DNA.
I inoculated 4 LB broths with single colonies from agar slopes E5 and E6. I placed 2 of these broths in a shaking incubator at 37ºC and 2 in a static incubator at 37ºC. These broths were incubated for 2 hours and then the OD for each was measured using a bench top spectrophotometer and un-inoculated LB broth as a reference. I found that incubating LB broth cultures for 4 hours in a shaking incubator at 37ºC achieved the best result and most appropriate OD.
Finally I used an Escherichia coli ATCC 25922 culti-loop to inoculate nutrient agar. This agar plate was incubated overnight at 37ºC.
Tuesday 1st April 2014
Today started with a training session regarding the QIAgen QIAcube. The QIAcube is a fully automated sample preparation workstation including integrated centrifuge, heated shaker, pipetting system, and robotic gripper, which can be used in the purification of DNA, RNA and proteins. It utilises QIAgen’s well established spin columns alongside QIAprep miniprep kits, eliminating the need for manual steps to achieve purification.
I have already manually extracted plasmid DNA using alkaline lysis from my Lincoln collection samples, however it is very time consuming and requires a lot of pipetting. The QIAcube can process 12 samples in around 50 minutes and although it gives less end product than alkaline lysis the final sample is a lot purer. As I mentioned last week I would like to re process all my Lincoln collection samples and re run the resulting plasmid DNA through gel electrophoresis. This is to ensure that all samples I take forward to PCR definitely have plasmid DNA and therefore can be assessed for the presence of CTX-M specific Bla genes.
I won’t write about every stage the QIAcube conducts, but this video is great if you are really interested! Sample preparation begins by pippetting 2ml of an overnight culture into 2ml eppendorfs and centrifuging for 5 minutes at full speed to produce a cell pellet (I love these 2ml eppendorfs! They save so much time when centrifuging 2ml overnight cultures). The supernatant is then tipped away and the resulting eppendorf containing the cell pellet placed into the QIAcube.
- Setup – Depending on what procedure you are conducting will depend how your spin column trays are set up, pipette types are used and buffers are required.
- Load Check – The QIAcube then ensures all the correct buffers, tips and spin columns trays are present as well as calculating how many you will need based on the amount of samples you have loaded.
- Resuspension, Lysis and Neutralise – Buffer is added to your centrifuged cell pellet and is shaken using the built in heated shaker.
- Bind – Lysates are added to the spin column within the integrated centrifuge, centrifugation allows collection of DNA/RNA/proteins.
- Wash – Wash steps are performed within the intergrated centrifuge.
- Elute – The remaining DNA/RNA/proteins are eluted through further centrifugation.
For my samples I used DNA miniprep, 5ml sample size and standard processing. Seeing as this was my first time using the QIAcube I found it really easy and simple to operate. The touchscreen moves through every stage you need to complete one at a time, from initial sample preparation to emptying the waste drawer.
Unfortunately I came across a few problems while trying to process my samples. If the QIAcube identifies a problem, it will stop and not continue from where it had reached. This can be a bit frustrating especially if there are several buffer solutions which are either unacceptable for use or too full/empty to complete the procedure. Half way through my first sample run, the QIAcube displayed an error code that was due to the robotic arm picking up the wrong tip. After checking inside the QIAcube it was noticed that the robotic arm had not picked up an incorrect tip, but instead was just disposing of tips without using them. This halted use and I decided to trouble shoot the problem tomorrow.
Finally I removed the overnight Escherichia coli ATCC 25922 nutrient agar plate and inoculated LB broth with a single colony. This broth was incubated overnight at 37ºC. I also inoculated a nutrient agar slope with a single colony and added to my on bench collection to grow slowly at room temperature.
Wednesday 2nd April 2014
Today started with referring to the QIAcube manual. After checking through the appropriate sections it was identified that the problem could be occurring for two reasons:
- Dirty tip sensor
- Defective O ring in robotic arm
To start with the tip sensor was cleaned and the procedure re run to see if this had fixed the problem. Unfortunately it did not so a tightness test was conducted to see whether the O ring within the robotic arm was working. This test immediately failed, and after observation through the QIAcube front window it was seen that a correct tip was selected, however it was repeatedly being hit against the tip sensor unit before being read. This was resulting in an incorrect tip message being displaced and was most likely due to a defective O ring, causing the tip to not be properly picked up by the robotic arm.
In order to change the defective O ring specialist kit was ordered from QIAgen, including QIA tip adaptor tools, ring replacement kit and rings. As soon as this equipment arrives in the lab we will be able to replace the defective O ring and get on with plasmid DNA purification!
I then removed the overnight Escherichia coli ATCC 25922 LB broth and used it to produce 4 glycerol stocks by combining 500μl of the broth and 500μl 10% LB glycerol. These samples were pipetted into screw top eppendorfs, labelled and stored in the freezer.
Finally I decided to continue producing cell pellets from my bacterial collection in preparation for plasmid DNA purification. This was conducted by inoculating LB broth with single colonies from my Lincoln Escherichia coli nutrient agar slope collection (E49 – 55). These broths were then shaken and incubated overnight at 37ºC.
Thursday 3rd April 2014
Today started with removing the overnight LB broths inoculated with further samples from my Lincoln Escherichia coli nutrient agar slope collection (E49 – 55). Sample preparation was conducted by pippetting 2ml of the overnight culture into 2ml eppendorfs and centrifuging for 5 minutes at full speed to produce a cell pellet. The supernatant was then tipped away and the resulting eppendorf containing the cell pellet stored in the freezer until it can be used in the QIAcube.
I then began new bacterial plug production through inoculating LB broth with single colonies from E.5 and Escherichia coli ATCC 25922. Based on incubation on Monday, these broths were incubated for 4 hours in a shaking incubator at 37ºC to achieve the best result and most appropriate OD.
Once an appropriate OD was 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.
Finally I continued producing cell pellets in preparation for plasmid DNA purification, as well as finishing off glycerol stocking the newest samples in my Lincoln collection. Again, this was conducted by inoculating LB broth with single colonies from my Lincoln Escherichia coli nutrient agar slope collection (E56-105). These broths were then shaken and incubated overnight at 37ºC.
Friday 4th April 2014
The week concluded with the washing of my new 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 run next week!
Finally I finished sample preparation and glycerol stocking as before, storing the final cell pellets and glycerol stocks in the freezer.
Now I have a new batch of bacterial plugs prepared I really want to run these using my previously established settings. Hopefully the equipment required to fix the QIAcube will also arrive next week so that I can get on with plasmid DNA purification. If not, then I plan to continue preparing for PCR, by working on a starting protocol using the journal I took my primer sequences from.
The week after next I will be taking a week holiday for Easter, after which I would really like to have started PCR, be finalising settings for PFGE, and begin collecting and processing samples from my second site, Bradford.
A Year in the Lab…