Plasmid loss test results:
MG1655 100% loss, dam 100% loss, rcsA 0% loss, crp 0% loss
Weird

Big M (gly stock) grew well on LB, only 1 colony on LB + amp. Huge colonies. Doesn't look like it's amp resistent.

DNA electrophoresis on pMOB45 and pkd46
1. Prepare (1%)agarose gel (dissolve 5g agarose powder in 500ml TAE buffer, instead of water because it's a better electrolyte)
2. Microwave 30s at a time until boil and everything dissolves and mixed well
3. When it is cooled to 60 degrees, add 10ul Ethidium bromide (EtBr)(will glow in UV when bound to DNA), be careful cos it's carcinogenic.
4. Set up frame, ensure that frame is horizontal with spirit level
5. Pour gel into frame until at least 1cm of well is formed, don't pour all the way to the top though or no wells will be formed, ensure that there are no bubbles in the area where DNA will pass through by pushing them to the side with paper, or it will hinder DNA movement
6. Leave for gel to solidify
7. Remove template carefully, bring solidified gel to electrophoresis machine(?), be careful not to drop the gel, it's slippery
8. Add enough 1xTAE buffer to cover the gel and the fill the wells
9. Pipette 3ul 6x dye onto parafilm (use the inner side) (1ul for 5ul of DNA), pipette 10ul DNA and 5ul 1kb DNA marker (2ul dye)
10. Mix DNA and dye with pipette
11. Suck up the mixture, make sure there's no air bubble, insert into one well
12. When all wells are filled, can start current. Make sure DNA runs from well towards red(+) terminal. V= 90V (fixed), I= variable, time = 30- 45min. Bubbles of H2 and O2 seen
13. After 30min, dye shows blue, violet, yellow bands, chromatography, cool
14. Remove frame with gel, be careful don't drop the gel, bring to UV (?)machine.
15. Put frame into machine, start UV, bands appear on computer, focus, adjust lighting, zoom, freeze and print. Or, put frame under UV light, beautiful bands.

Promega Miniprep plasmid extraction
Centrifugation Protocol
Production of Cleared Lysate
1. Pellet 1–10ml overnight culture for 5 minutes.
2. Thoroughly resuspend pellet with 250µl Cell Resuspension Solution.
3. Add 250µl Cell Lysis Solution to each sample; invert 4 times to mix. (solution should clear a bit)
4. Add 10µl Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature. (murky solution becomes clear as proteins and endonucleases are broken down)
5. Add 350µl Neutralization Solution; invert 4 times to mix. (ppt formed)
6. Centrifuge at top speed for 10 minutes at room temperature. (cell wall, proteins, chromosomal DNA enters residue(?))

Binding of Plasmid DNA
7. Insert Spin Column into Collection Tube.
8. Decant cleared lysate into Spin Column.
9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough and reinsert Column into Collection Tube. (plasmid bind to spin column filter)

Washing
10. Add 750µl Wash Solution (ethanol added). Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube.
11. Repeat Step 10 with 250µl Wash Solution.
12. Centrifuge at top speed for 2 minutes at room temperature.

Elution
13. Transfer Spin Column to a sterile 1.5ml microcentrifuge tube.
14. Add 100µl of Nuclease-Free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature. (DNA enters and dissolves in water)
15. Discard column and store DNA at –20°C or below.

Summary
Centrifudge overnight culture to get cell pellet.
Remove culture media.
Resuspend cells.
Lyse cells.
Neutralize.
Insert column in Collection Tube.
Transfer cleared lysate and bind DNA.
Wash, removing solution by centrifugation.
Elute plasmid DNA.

DNA quantification (Gene quant)
1. Wavelength = 320, follow instructions
2. Insert blank (water)
3. Use capillary tube to suck up 1cm of DNA + water
4. Poke in plasticine to stopper the end
5. Put into spectrometer, read, note down conc(ng/ul), 260/280 value (should be around 1.8, measures protein contamination), 260/230 value to show purity of sample

Plated bigM, NS3, MG1655 on EMB agar
Plated bigM on LB and LB+ amp, inoculated bigM in LB and LB+ amp shake flasks
Incubate at 37degrees overnight