Team:Victoria Wellington/Notebook
Team
Victoria
Notebook
April
Cell Competency 18/04/2019
2x 400mL flasks with 1 in 100 dilution of overnight culture. Incubate at 37°C.
Grow until OD600 reaches 0.4-0.6.
Pour 1 flask worth of culture into pre-chilled 50mL Falcon tubes and ice for 20 minutes.
Spin at 3190rpm for 10 minutes, decant supernatant and add remaining culture on top of pellets.
Spin again
Add 50mL MQ over 4 tubes and resuspend pellets. Tip resuspended cultures into remaining 4 Falcon tubes and resuspend. Spin at 3190rpm for 10 minutes. Remove supernatant.
Add 50ml 10% glycerol to 2 tubes and resuspend pellets (down to 2 tubes). Spin at 3190rpm for 10 minutes.
Add 50mL to 1 tube, resuspend pellet, tip over remaining tube and resuspend pellet at 3190rpm for 15 minutes.
Tip off all supernatant. Allow residual glycerol drip down sides aand add additional 400mL of 10% glycerol. Fully resuspend final pellet with 1mL tipe. Should have ~1.1mL of super dense culture.
Freeze 85mL aliquots at 80°C.
Snap freeze in freeze block and test competency from frozen.
This was done using two E. coli strains: DH5α and BL12.
Carbon Paste Experiment 26/04/2019
Aim: Build electrodes of different graphite to oil ratios. Test their conductivity in salt solution
Method:
1. 2g of graphite was ground with 2mL of paraffin oil.
2. 1g of graphite was ground in a mortar and pestle with 2mL of paraffin oil.
Observations: The 1:1 ratio was a thick viscous paste. The 2:1 was near liquid in viscosity. It look like the 2:1 will not be able to support itself in the anode.
3. Not enough paste was obtained. So 4g of graphite to 4mL of paraffin was used for the 1:1 anode. 3g of graphite to 6g of paraffin was used for the 1:2 anode
Observations: The 1:2 anode was liquid and ran straight through. The 1:1 paste was a good consistency and could hold itself & wires in the tube.
4. 12g of NaCl was added to 100mL of distilled H2O to make a saturated NaCl (aq) solution.
5. A circuit was set up as below
6. The LED glowed proving the paste was conductive
Conclusion: From this experiment we proved that the electrodes were conductive and that 1:1 paste is a very good electrode!
However we found some issues trying to take accurate data past that.
Firstly, trying to fix the wire in place proved to be difficult. Due to this, we couldn't calculate the resistance of the CPE as the system would always be disturbed upon us plugging in the multimeter
In order to calculate the resistance we will need some way to immobilise the wire (using wax most likely) to get an accurate reading from the meter
May
PCR (+ Prep) 01/05/2019
Alex prepared some agar, made more broth.
Made up 1x TE buffer.
Add 250µL to primers to bring up concentration to 100 pmol/µL.
Primers at 100 pmol/µL. Need to dilute further to be 0.5 µM = C = 0.5x10-6M
Currently:
C = 100 x 10-12 mol/µL-1.
C = 100 x 10-6 mol/L-1.
Dilution factor:
C2 = 𝑥/50µL x C1
0.5x10-6M = 𝑥/(50x10-6L)x 100x10-6M
𝑥/50x10-6L = 5x10-3
𝑥 = 2.5x10-7L
𝑥 = 0.25µL of primer
PCR Method (from Alistair):
Reaction:
2x master mix --> 10µL
10 fold diluted forward primer --> 1µL
10 fold diluted reverse primer --> 1µL
10 fold diluted template --> 1µL
7 µL PCR H2O
Method:
5 µL of forward primer was diluted in 45 µL of PCR H2O.
The same was done for the reverse primer.
5µL of template is added to 45 µL of PCR H2O.
Add all reagents minus template to PCR tubes.
Add template last, place in machine.
Miniprep of Plasmid 02/05/2019
Plasmid needs to be mini-prepped from bacteria for PCR
Method:
2 mL of cells were added to an Eppendorf tube (we had 4 tubes).
The tubes were centrifuged and liquid poured off.
Note: 2 mL can't fit into one Eppendorf tube. So add 1 mL, centrifuge, pour off unwanted liquid and then add 1 more mL.
Centrifuge at full speed for 1 minute.
Suspend pellet in 200µL of PDI.
Vortex the cells to mix thoroughly.
Add 200 µL of PD2.
If SDS is insoluble in PD2 (you can see crystals) add the bottle to a hot water bath for 5 minutes.
Invert the tube 4-6 times to mix. Note: after PD2 is added be gentle as PD2 makes DNA single-stranded so
it's now susceptible to mechanical damage.
Wait for 2 minutes.
Add 300µL of PD3.
Centrifuge for 5-10 minutes.
Pipette the clear liquid into Geneaid miniprep column (Plasmid sticks to the column).
Spin for 60 seconds
Pass the flow through back through the column and spin for a further 30 seconds.
Discard flow-through, return the catcher.
Add 400µL of W1, spin for 60 seconds and discard the flow-through.
Add 2x 300µL of wash to column. Spin for 60 seconds each time. Discard flow-through.
Spin for 2 minutes to remove CH3CH2OH.
Transfer column to fresh Eppendorf.
Add 100µL of 65°C EB as this removes DNA from column.
Let stand for 1-5 minutes.
Column can also be heated to 65°C to enhance elution.
Centrifuge for 1 minute.
Harvest DNA!
Initial nanodrop = 6.6ng/µL DNA.
Second/Refined = 13ng/µL DNA.
PCR 06/05/2019
Dilute primers 10-fold from 100µM to 10µM.
Used four PCR tubes, 1 control and 3x pET 28a +
In each reaction vessel:
20 µL Q5 master mix
1 µL Forward primer
1 µL Reverse primer
1.2 µL Template (plasmid)
Total reaction volume (µL) = 23.2
Agarose Gel Electrophoresis
Gel composition:
100 mL 1% agar mix
5 µL of ethidium bromide
Run for 30 minutes at 135 volts
June
1/6/2019
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Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.
Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.
2/6/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
July
1/7/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.
Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.
2/7/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
August
1/8/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.
Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.
2/8/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
September
1/9/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.
Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.
2/9/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
October
1/10/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.
Donec tristique aliquet orci, non ultrices tellus varius in. Sed ut varius quam. Duis porta orci sit amet enim placerat, sed vestibulum elit placerat. Sed mattis pharetra consequat. Etiam eleifend non lectus vel semper. Sed sodales maximus odio eget cursus. Nulla et metus diam. Fusce elementum sodales lacus, sed ultrices est laoreet a. Mauris sollicitudin magna in massa malesuada facilisis sed sit amet ligula. Quisque nec pulvinar sapien. In nec imperdiet nunc. Sed erat nisl, vehicula vitae auctor in, suscipit in nunc. Quisque rhoncus vel nibh sit amet tincidunt. Quisque condimentum consectetur porttitor. Proin odio nisi, interdum fermentum porttitor in, vehicula non urna.
Vestibulum ultrices, massa non dictum ornare, mauris turpis efficitur mauris, eget placerat odio lacus vitae neque. Maecenas sem tellus, semper nec orci at, imperdiet lobortis metus. Mauris vel blandit diam. Sed tincidunt, enim et porttitor lobortis, ligula ipsum egestas lorem, in pellentesque sapien turpis a sem. Quisque sollicitudin libero in ante mattis, in interdum libero cursus. Duis mollis neque eu sem eleifend, et venenatis libero maximus. Cras ultricies diam elit, at vehicula dui consectetur sit amet. Fusce in tempus eros.
2/10/2019
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Phasellus luctus pulvinar lacinia. Donec bibendum at ex ac viverra. Donec lorem nisl, vehicula nec justo ut, rutrum dictum ex. Quisque mauris risus, sollicitudin in dapibus a, congue eget mi. Ut et fringilla metus. Donec turpis justo, vehicula quis ex at, scelerisque semper libero. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Proin lobortis quam quam, sed tincidunt lorem mattis a. Phasellus nec ornare tortor, eu dictum elit.