- Maxi-preped parts 1I and 6B.
- 6B to replaced used up maxi-prep stocks
- 1I maxi-preped in order to use for ligation. (Part 2H discovered to be faulty on Wednesday).
- Minipreped J37016 R colonies 1, 2, 3, 4
- Gels show that colony 2 and 4 were successful
- Cultured up colony 2 for maxiprep tomorrow
Cultured up parts 12D and 5M in order to maxi first thing tomorrow.
- Made up 2 batches of 200 mL M9
- 40 mL 10x M9 salts
- 12 mL 10 mg/mL Thiamine
- 4 mL 40% glycerol
- 8 mL 10% casamino acids
- 8 mL 0.1M MgSO4
- 80 μL 0.5 M CaCl2
- Make up to 400 μL with sterile water
- Filter sterilise the entire batch (see Sue)
- Kept in fridge for use
- Made up 2 batches of 500 mL LB
- 10 g Tryptone
- 5 g Yeast Extract
- 10 g NaCl
- Make up to 1 L with distilled water
- Take to autoclave
To do tomorrow
- 4G - 2 colonies
- 1M - 2 colonies
- J37015RS - 4 colonies, more colonies to ensure we have the final construct that we want, then take to sequence if we do
- 2H (2nd attempt) - 2 colonies, to check if the DNA is actually wrong in the parts registry
- J37016, then take to sequence
- 12D - missing maxiprep???
- 5M - also missing maxiprep???
- Please find the concentrations of the DNA by taking to Dr. Jensen on 5th floor of biochem building
For sequencing - for tomorrow
- Use total of 12.5 uL in a 0.5 mL eppendorf tube
- 1 μg of DNA - you will need the concentrations
- 12.5 pmol primer
- Make up to 12.5 μL with ultrapure water
- Take to either Dr. Mann or Dr. Jensen to sequence
- Because we have established that there is a problem with the Spe1 restriction site on our 2H culture and as this is now holding up our progress, we have decided to change our ligations steps. It will increase the number of ligations we are doing by one, but the time that this takes will not be increased, as we are able to do two of these ligations in parallel.
- Tomorrow we will ligate parts 12D and 9G together and parts 1I and J37019 together
- (1I + 9G are the components that 2H is made up of)
- We can then ligate the two ligated parts together.
- If all goes to plan this test construct will be complete by this time next week
J37020 alternative construction
- J37031 = J37019 + B0015 (1I), pLuxR + RBS + LuxR + Terminator
- J37019 (insert) cut with EcoRI & SpeI, expected size = 862 bp
- B0015 (vector) cut with XbaI & EcoRI, expected size = 129 bp + 3189 (pSB1AK3) = 3318 bp
- J37032 = R0062 (9G) + I13504 (12D), pLuxR + RBS + GFP + Terminator
- I13504 (insert) cut with XbaI & PstI, expected size = 875 bp
- R0062 (vector) cut with SpeI & PstI, expected size = 55 bp + 2079 bp (pSB1A2) = 2134 bp
- J37020 test construct = J37031 + J37032
- J37031 (insert) cut with EcoRI & SpeI, expected size = 999 bp
- J37032 (vector) cut with XbaI & EcoRI, expected size = 938 bp + 2079 bp (pSB1A2) = 3017 bp
- J27020 miniprep, expected size = 1945 bp (part length) & 2079 (pSB1A2)
Experiments abandoned due to contaminated M9 media re-ordering Should repeat tomorrow