Team:Edinburgh/Notebook/HeroinPurity

InterLab Notebook

29/06
Take BBa_K823005, BBa_K823008 and BBa_K823013 out of the distribution kit and transform into Top10.

30/06
Inoculate cells from single colonies on the transformation plates into 10ml LB with 10µl of relevant antibiotic. Transform BBa_I13504 into Top10.

01/06
Make glycerol stocks of cultures. Miniprep cultures from 30/06. Nanodrop elute.

Inoculate BBa_I13504 transformants.

02/07
Miniprep cultures using QIAprep spin Miniprep kit and nanodrop.

23/07
Make fusions through biobrick assembly. Digest BBa_K823005, BBa_K823008 and BBa_K823013 at 37ºC for 1.5 hours.
Promoter digestions:
4 µl DNA (25 ng/µL)
0.1 µl SpeI
0.1 µl PstI
1 µl Buffer 2.1
2.8 µl H₂O


Digest BBa_I13504.
GFP digestion:
4 µl DNA (30 ng/µl)
0.1 XbaI
0.1 PstI
1 µl Buffer 2.1
2.8 µl H₂O

Treat promoters with Antarctic Phosphatase: 0.2µl phosphatase, 0.8 µl buffer for 15 min at 37ºC. Heat inactivate at 80ºC for 20 min.

Ligate GFP and promoters together.

2.3 µl promoter
4 µl GFP
1 µl T4 ligase buffer
0.5 µl T4 ligase
1.8 µl H₂O

This includes 4 ligations as there is a control ligation for each promoter with extra H₂O instead of insert.
Run the ligation at 16ºC for 1 hour. Heat inactivate ligase at 65ºC for 10 min.
Transform ligations into Top10 chemically competent cells.

24/07
Plates were placed in cold room at 4ºC.

26/07
Inoculate colonies on transformation plates in a 50ml Falcon tube with 10ml LB and 10µl 1000x chloramphenicol. No growth on controls.

27/07
Make glycerol stocks. Miniprep using QIAprep spin Miniprep kit and nanodrop.

Diagnostic digest:
2 µl Cutsmart
0.25 µl NotI
100-500 ng DNA
up tp 20 µl H₂O
PICTURE IN HERE

Sequence confirm BBa_K823005 + BBa_I13504 1, BBa_K823005 + BBa_I13504 2, BBa_K823008 + BBa_I13504 1 and BBa_K823008 + BBa_I13504 2.
BBa_K823005 + BBa_I13504 1 Sequence Alignment:

BBa_K823008 + BBa_I13504 1 Sequence Alignment:

28/07
Retry BBa_K823013 + BBa_I13504 fusion. Run the digestions at 37ºC for 1.5 hours.
Promoter digestions:
4 µl BBa_K823013 (25 ng/µL)
0.1 µl SpeI
0.1 µl PstI
1 µl Buffer 2.1
2.8 µl H₂O

Digest BBa_I13504.
GFP digestion:
4 µl BBa_I13504 (30 ng/µl)
0.1 XbaI
0.1 PstI
1 µl Buffer 2.1
2.8 µl H₂O

Treat BBa_K823013 with Antarctic Phosphatase: 0.2µl phosphatase, 0.8 µl buffer for 15 min at 37ºC. Heat inactivate at 80ºC for 20 min.
Ligate the two parts together at 16ºC for an hour.
2.3 µl BBa_K823013 digest
4 µl BBa_I13504 digest
1 µl T4 ligase buffer
0.5 µl T4 ligase
1.8 µl H₂O
Heat inactivate the ligations (one without insert) at 65ºC for 10 minutes.
Transform the ligation into chemically competent Top10s.

29/07

Inoculate two colonies from the transformation plate into a 50ml Falcon tube with 10ml LB and 10µl 1000x chloramphenicol. No growth on control plates.

30/07
Make glycerol stocks. Miniprep cultures using QIAquick spin and Miniprep Kit. Nanodrop elute.


Diagnostic digest:
1 µl DNA
0.25 µl NotI
2 µl Cutsmart
16.75 µl H₂O PICTURE IN HERE

Sequence confirm BBa_K823013 + BBa_I13504 3 and BBa_K823013 + BBa_I13504 4.

05/08
Get the positive and negative controls out of the distribution kit. Positive control: BBa_I20270. Negative control: BBa_R0040. Transform into chemically competent Top10s.

06/08
Inoculate two colonies from each transformant plate into 50ml Falcon tubes with 10ml LB and 10µl 1000x chloramphenicol. No growth on transformation control plate.

07/08
Miniprep cultures using QIAquick Spin and Miniprep Kit. Nandrop elute.

Diagnostic digest:
1µl DNA
0.25µl NotI
2 µl Cutsmart
16.75 µl H₂O
PICTURE IN HERE


12/08
Re-transform BBa_I20270 and BBa_R0040 from the distribution kit into chemically competent Top10s.

13/08
Inoculate two colonies from each transformation in a 50ml Falcon tube with 10ml LB and 10µl 1000x chloramphenicol.

14/08
Make glycerol stocks. Miniprep using QIAquick Spin and Miniprep Kit. Nanodrop.



17/08
Diagnostic digest:
1µl DNA
0.25 µl NotI
2 µl Cutsmart
16.74µl H₂O
PICTURE IN HERE

Sequence confirm.

BBa_K823013 + BBa_I13504 1 Sequence Alignment:


24/08
Re-streak all of the parts onto fresh LB+chloramphenicol plates.
25/08
Inoculate one colony from each of the plates into 50ml Falcon tube with 10ml LB and 10µl 1000x chloramphenicol.