Team:Newcastle/Project/Labwork/Week1/Week1

=Introductory Lab Sessions Week 1= (30th June - 3rd July 2009)

Outline for Week 1 - 30th June to 3rd July

 * Tuesday
 * 1) Find the strain
 * 2) If plates are unavailable, the steps for preparing plates are:
 * 3) Cool the media to approximately 50 to 60 degree Celsius
 * 4) Fill the plate half full
 * 5) Dry them
 * 6) Get the strain from the freezer
 * 7) Streak the plates
 * 8) Incubate the plates
 * 9) Prepare other solution and medias needed
 * Wednesday
 * 1) From the plates which were streaked and incubated on Tuesday, pick up a single colony using a loop
 * 2) Note: When looping, flame the loop till it is cherry red to reduce the risk of contamination
 * 3) Inoculate the colony with 3ml of LB
 * 4) Incubate the media in the incubator shaker at 37 degree Celsius to be used on Thursday
 * 5) Note: The tubes containing the media should be tilted at an angle for optimal growth conditions
 * Thursday
 * 1) Prewarm the media at 37 degree Celsius
 * 2) Sample->wash 2x-? Suspend in 1ml->freeze->-80 degree Celsius
 * Friday
 * 1) Transformation test with a plasmid

Lab session: 30th June

 * We got samples of E.coli DH2 Alpha strains and placed them into three plates. They are placed for incubation at 37°C
 * Glycerol concentration will need be changed as 50%
 * We have created 0.1M of CaCl2 using 5.5 gram of CaCl2. The final solution is 0.5 liter. We placed them into 4 boxes (three 100ml and one 200ml). They have blue covers. We have left them for autoclaving.
 * We prepared LB solution using 20gr LB. Th final volume is 1 liter. We put them into boxes and left for autoclaving.

Lab session: 1st July
Things we need for today:
 * LB
 * Containers
 * Sterile tubes
 * Space in incubator for tonight


 * We plated out single cultures from yesterday's plates for a practice.
 * We inoculated 2ml of LB with single cultures from yesterday's plates and put them in the shaking incubator.
 * We made up some 50% glycerol and it was sent to be autoclaved.

Things we need for tomorrow (Thursday):
 * Have we used up a lot of anything- is there enough for Thursday?
 * 1) Sterile tubes
 * 2) Pipette tips
 * 3) Solutions

Culture
 * 1) Sterile flasks for culture (500ml)
 * 2) Spectrophotometer and cuvettes (where)
 * 3) Arrange space in shaking incubator

Harvest cells
 * 1) Centrifuge/ centrifuge vial (200ml?)
 * 2) Ice bucket and ice
 * 3) Glycerol
 * 4) Sterile microfuge tubes
 * 5) Liquid nitrogen

Lab session: 2nd July

 * 1ml of preculture (which had been left overnight) was taken and used to inoculate 200ml of LB (standard bacterial broth). The flask which contained the mixture was then placed into the shaker, which both incubated the culture at 37 degrees Celsius and constantly shook the flask.


 * Once 2 hours had elapsed, the optical densities of the cultures were taken using a spectrophotometer. This was to determine whether the cells had grown and if they had, where the cells were in their growth phase. For competency, E.coli need to be in early log phase. The results were as follows:


 * The next step was to transfer the three cultures into a centrifuge and allow them to spin for 10 minutes, at a speed of 8,000 rpm and at a temperature of 4 degrees celsius. Because balance is required in a centrifuge (i.e. even numbers), we only used two of the tubes.


 * Once spun, the supernatant was removed and the pellet was resuspended in calcium chloride, which had been placed in a fridge since the solution was made. The concentration of the calcium chloride was 0.1M and the volume was 40ml.


 * The cells emersed in the calcium chloride were then held in ice for 40 minutes.


 * Once 40 minutes was complete, the cells were once again centrifuged at a speed of 8,000 rpm and at the temperature of 4 degrees celsius. After being spun, the supernatant was removed and the pellet was resuspended in 1ml calcium chloride again. This solution was again pre-chilled in the fridge, was again 0.1M but only 1ml was applied this time round.


 * 50% glycerol was then applied to the mixture. Because the concentration of the glycerol to cell mixture was supposed to be 10%, we applied 0.ml to 0.9ml of cell mixture.


 * The resulting mixture was then placed into 50 microfuge tubes (each with a capacity of 100 microlitres) with these tubes being placed into a flask of liquid nitrogen. This process shock-freezes the E.coli cells.


 * After being given a few minutes to confirm freezing of the cells, the tubes were immediately scooped out of the flask and transferred immediately into a -80 degrees celsius freezer for storage.

Lab session: 3rd July
Today we were transforming DNA using Phil's protocol, however we used the following changes:


 * We added 3µl of DNA to our cells.
 * We plated out 100µl of our dilutions.
 * We incubated our transformed cells for the full 60 mins.
 * We used the ampicillin resistance plasmid '435'

Procedure

 * We followed the protocol using sterile technique where possible, as well as wearing gloves to prevent contamination of the DNA.
 * Glass tops such as LB jars can be run under the flame, however plastic eppendorfs cannot so working around the flame is sufficient.
 * We found it easier if the person pipetting held the eppendorf as they can see what they're doing better.
 * Prof. Aldridges lab uses sterile beads to plate ourt rather than plastic spreaders, these are a bit fiddly so it may take a while to get used to tipping the right amount out!!

Our serial dilutions were prepared as follows:

 * Using the waiting times within our procedure we set up 6 labelled eppendorf tubes with 900µl of LB, ready for the series dilution.
 * We took 100µl of our incubated cells and put them in tube 1, mixed the solution briefly and then proceded to take 100µl of the diluted cells and tranfer them along the line of eppendorfs, resulting in eppendorfs containing 10-1 to 10-5 solutions for both the control and 'transformed' cells.
 * We plated all 6 transformed E.coli solutions onto LB+amp agar plates, and the controls we plated onto both LB+amp plates(to check for contamination) as well as plain LB plates (in order to later calculate transformation frequency).