Team:Newcastle/Project/Labwork/Week1/Week4

=Introductory Lab Sessions= (23rd July - 24th July 2009)

The fourth week initial planning, 23rd July - 24th July

 * Thursday
 * Friday

Lab session: 23rd July
Please note that all of the tasks written in purple play a part in the real project lab work - they are not introductory lab session work. They just happen to run parallel to some of the introductory lab sessions this week

Introduction
In the last session of last week’s set of practicals, we had attempted to conduct a midi-prep of the plasmids containing the RFP and the GFP genes (derived from E.coli DH5alpha cultures 2 and 3 respectively). However we were unsuccessful in loading the two prepared plasmids into the gel in our electrophoresis attempt. When inserted into the wells the DNA immediately floated and dispersed within the running buffer of the gel electrophoresis apparatus.

The reason for this anomalous result was that the samples still contained a high concentration of ethanol, which should have been completely washed away prior to loading into the wells. Washing solution 2 contained ethanol as an ingredient. After analysis of the situation, a few conclusions were drawn:


 * For the washing process 15ml falcon tubes should have been used instead of 50ml falcon tubes.
 * The centrifugation step should have been allowed to run for longer to ensure all of the wash solution had passed through the buffer column.

However all was not lost; the plasmid DNA was still there and in usable form. The challenge was how we could extract it successfully.

The answer to our problem is ethanol precipitation. By precipitating the ethanol from our DNA sample, we could then process the plasmid DNA and run on the gel as usual.

In the meantime we also need to grow up cultures containing the plasmid backbone pMUTIN4, which will allow us in the near future to transfer our chosen BioBricks from the E.coli cells we are working with into Bacillus subtilis.

Another task which we have to complete is to prepare the BioBricks which are needed for our system and are found in the Spring Distributions.

Practical Outline

 * 1) Precipitate the ethanol from our DNA plasmids (containing the RFP and GFP genes) and analyse them through DNA gel electrophoresis.
 * 2) Grow up some DH5alpha E.coli cells containing the pMUTIN4 plasmid vector
 * 3) Hydrate and prepare BioBricks present in Spring Distributions.

Ethanol Precipitation
We used the ethanol precipitation protocol and carried out these steps:
 * Added 0.2ml of sodium acetate (a tenth of the volume of DNA) to 2mls of DNA from both GFP and RFP samples.


 * To these mixtures, 6.6ml of 100% ethanol was added.


 * The 8.8ml of GFP DNA + sodium acetate + ethanol solution was then divided into 8 Eppendorf tubes – each Eppendorf tube containing 1ml of solution.


 * In the same way, the 8.8ml of RFP DNA + sodium acetate + ethanol solution was then divided into 8 Eppendorf tubes – each Eppendorf tube containing 1ml of solution.


 * Both sets of Eppendorf tubes were then stored in the fridge for further work tomorrow.

Preparing cultures containing pMUTIN4
'''For this exercise 2 x 500ml flasks were used. Into each flask 100ml of LB solution was added (under aseptic conditions) and also to each flask 0.5ml of ampicillin was added (ampicillin resistance is conferred by the pMUTIN4 plasmid). A sample was then taken from an agar plate containing E.coli possessing pMUTIN4 and was added to each of the two flasks. The 500ml flasks were then left in the 37ºC shaking incubator overnight.'''

Preparing the chosen BioBricks
'''There are five BioBricks which are likely to be used by our system. They are:''' '''Each well containing these BioBricks was firstly identified and marked with a pen (in the corners of the wells). Once we had confirmed that the correct wells had been identified, a pipette containing 15ml of distilled water was then used to pierce the film lids of the wells and hydrate the DNA within. After a few rounds of mixing the DNA inside the wells, the BioBricks were extracted and placed into Eppendorf tubes which were transferred to the -20ºC freezer for storage.'''
 * 1) BBa_C0056 – cI repressor (lambda phage)
 * 2) BBa_B1002 – Terminator sequence
 * 3) BBa_C0077 – CinR activator
 * 4) BBa_C0076 – Autoinducer synthetase
 * 5) BBa_R0077 – Promoter (CinR and HSL regulated, RBS+)

'''The locations of the BioBricks can be found here – all are contained within the iGEM spring distributions. The hyperlinks for each BioBrick below will direct you to the distribution information page:'''
 * 1) BBa_C0056 -  2009 kit plate 1 – well 24A – plasmid pSB1A2
 * 2) BBa_B1002 – 2009 kit plate 1 – well 4B – plasmid pSB1AK3
 * 3) BBa_C0077 – 2009 kit plate 1 – well 14F – plasmid pSB2K3
 * 4) BBa_C0076 – 2009 kit plate 1 – well 14D – plasmid pSB2K3
 * 5) BBa_R0077 – 2009 kit plate 1 – well 10K – plasmid pSB1A2

Introduction
As far as the introductory lab sessions go, we have completed one half of the ethanol precipitation procedure; once completed the procedure will see the plasmid pSB1A2 (containing GFP) and the plasmid pSB1AT3 (containing RFP) free from ethanol. This means they can be loaded onto gel and analysed appropriately (i.e. DNA gel electrophoresis)

Practical Outline

 * 1) Remove the Eppendorf tubes from fridge and resume the rest of the ethanol precipitation protocol.

Ethanol Precipitation continued
So far we have added sodium acetate to the 2 DNA samples and also added ethanol. The team have also divided the samples into Eppendorf tubes and stored them in the fridge overnight (a replacement for the15 minute incubation-on-ice step). The following was carried out:
 * The 16 Eppendorf tubes (8 containing GFP and 8 containing RFP) were spun down for 20 minutes at 13,000 rpm.
 * With the supernatant removed the 16 samples were rinsed in 70% ethanol and spun for 15 minutes. The supernatant was discarded (making sure that the DNA pellet was it disturbed) and the tube dried using the Speed Vac machine.
 * The DNA pellets were then resuspended in 50ul of distilled water and stored away in the -80ºC freezer.

Conclusion
Both the pSB1A2 plasmid containing GFP and the pSB1AT3 plasmid containing RFP will be analysed by DNA gel electrophoresis on next Monday’s lab session.