Introductory Lab Session – 16th July 2009
Introduction
In yesterday’s lab session, we prepared two chosen cultures of E.coli cells (one culture containing RFP and the other culture containing GFP) for midi-preps by inoculating 2 tubes of 3ml LB with them (2 different tubes for the two colonies). The cultures were then left overnight to grow under incubation; if the E.coli cells have grown successfully they will be midi-prepped today. If not, another attempt will be required. If the midi-preps are successful then analysis of DNA by gel electrophoresis will commence.
Practical Outline
Arun resuspending
E. coli containing RFP in 4ml of Resuspension + RNAse Solution
- If inoculations successful then carry out midi-preps on the 2 cultures of E.coli – one culture should contain GFP and the other should contain RFP.
- Analyse midi-prepped DNA through gel electrophoresis.
Observations
When the flasks were taken out of the orbital incubator, not only could it be seen that the LB solution had gone cloudy with the presence of E. coli cells but that the cultures were also brightly coloured red and yellowy green (for the RFP and GFP-containing E.coli cultures respectively). With this success, the two inoculated LB solutions were transferred to 2 falcon tubes (50ml) for the midi-prep procedure.
Procedure
Midi-prep
James resuspending
E.coli containing GFP in Resuspension (plus RNAse) solution
Arun holding falcon tube containing
E. coli + RFP just after lysis solution has been added! The colour will disappear completely upon inversion
The syringe barrel containing a culture of
E.coli after Resuspension Solution, Lysis Solution and Binding Solution have been applied
To carry out the midi-prep procedure, the team used Sigma-Aldrich’s GenElute Midi-prep kit and with it, the enclosed protocol. We chose to follow the Spin-method and carried out steps 1-6 and 1b to 11b missing out the DNA concentration step and also missing out DNA quantification. This is a summary of the steps:
- The two falcon tubes, each with 50ml of E.coli culture, were spun down for 10 minutes (under 4,500g); once centrifugation had finished the supernatants were discarded. To the pellets 4ml of resuspension solution (with RNAse included) was added and used to resuspend them.
- 4ml of lysis solution was applied to both samples (with both tubes inverted gently 8 times) and left for 5 minutes. As soon as this time had expired, 4ml of neutralisation solution was added. The 2 falcon tubes were gently inverted 6 times for mixture.
- 3ml of binding solution was added to the two solutions. Once the two falcon tubes were inverted twice, the solutions were placed into separate syringe barrels. The two culture solutions were allowed to remain in the barrel for 5 minutes.
- During this 5 minute waiting step, 4ml of Column Preparation solution were added to two Binding Columns, with the columns centrifuged for 5 minutes. When finished the eluate was removed.
- Half of the two culture solutions were syringed into the two binding columns. The columns were then centrifuged for two minutes and the solution eluted removed. The remainder of the two solutions was then placed into the binding columns and further centrifuged for 2 minutes. Like before the eluate was discarded.
- 4 ml of Wash Solution 1 was then added to both Binding Columns, which were then centrifuged for 2 minutes under 3,000g. The eluted solution was then discarded and to the 2 empty Binding Columns, 4ml of Wash Solution 2 was added. The columns were then centrifuged for 2 minutes again with the eluate removed.
- The binding columns were then transferred to clean collection tubes and to each column, 1ml of Elution Solution was added. The columns were then centrifuged for 5 minutes under 3,000g. THIS TIME THE ELUATE WAS RETAINED AND THE BINDING COLUMNS DISCARDED!
DNA Gel Electrophoresis
Neil loads DNA samples into gel but they disperse in buffer immediately
With the two midi-prepped plasmids prepared (midipreps of plasmid containing RFP and plasmid containing GFP) we were in a position to carry out DNA Gel Electrophoresis. Samples for electrophoresis were prepared in accordance of the protocol, i.e. 9ul of DNA + 1ul of loading buffer. The samples were then loaded onto the gel.
However as the samples were pipette just above the wells of the agarose gel they dispersed into the surrounding buffer solution; they did not fall into the wells! Something within the midi-prepped plasmid solution was causing them to float. It became clear that not all of the ethanol had been removed from the samples (the ethanol coming from Wash Solution 2).
In light of this incident, no gel electrophoresis could be carried out. Next week, we will have an analysis of the situation along with a solution to the problem.
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