Plates labelled:
Tetr_RBS + cyc_Term
Tetr_RBS + HAS_Term
OmpA + SacB_Term
Aseptically discarded 600μl solution from each eppendorf. Pipette mixed remaining and spread over plates.
5.05pm
Plates in incubator, 37°C overnight.
**Tuesday 04/09/12**
9.00am
Checked plates. Colonies have formed on all. Our own competent cells appear to be more efficient than first expected, less should be spread on future plates to make single colonies easier to pick.
These were all placed in the fridge, 4°C.
3.25pm
Plates out of fridge, read to make liquid broth.
4x made per plate – aseptic technique
Used 10ml broth
10μl Kanamycin
Scraped off single colony using pipette tip which was then ejected into liquid broth.
Tetr_RBS + HAS_Term and OmpA + SacB_Term had red and white colonies on the plates. Only the white colonies were selected.
4.20pm
Put into horizontal shaker set at 37°C, 220rpm – left overnight.
**Wednesday 05/09/12**
9.00am
Liquid broth aseptically transferred to new falcon tubes – leaving pipette tip behind.
10.00am
These were then centrifuged at 3900rpg for 10 minutes, 4°C.
The same protocol was followed as in the previous week for mini preps. However 40μl of clean water was added, instead of the previous 50μl, to boost the concentration of DNA in each sample.
1.30pm
Sample |
Conc of DNA |
DNA required |
Water required |
Tetr_RBS + Cyc_Term |
ng/μl |
μl |
μl |
1 |
136.8 |
3.65 |
13.15 |
2 |
154.4 |
3.24 |
13.56 |
3 |
126.5 |
3.95 |
12.85 |
4 |
114.8 |
4.36 |
12.44 |
Tetr_RBS + HAS_Term |
ng/μl |
μl |
μl |
1 |
68.4 |
7.31 |
9.49 |
2 |
65.3 |
7.66 |
9.14 |
3 |
100.6 |
4.97 |
11.83 |
4 |
92.8 |
5.39 |
11.41 |
OmpA + SacB_Term |
ng/μl |
μl |
μl |
1 |
123.6 |
4.05 |
12.75 |
2 |
116.7 |
4.28 |
12.52 |
3 |
250.1 |
2.00 |
14.8 |
4 |
95.7 |
5.22 |
11.58 |
2.45pm
Currently no enzymes, all DNA into freezer.
4.30pm
Streak plates made from the single colonies that appeared to have worked. Colonies that form should therefore also work.
All into incubator at 37°C overnight.
**Thursday 06/09/12**
3.30pm
Digestion
Digestion |
DNA |
500ng |
BUFFER (10x) |
2μl |
BSA (100x) |
0.2μl |
Water |
up to 20μl total V |
ENZYME 1 - ECORI |
0.5μL |
ENZYME 2 - PstI |
0.5μL |
Into incubator for overnight digestion.
**Friday 07/09/12**
11.00am
Gel electrophoresis run - insert image here
Sequences confirmed for:
Single genes confirmed for: