Team:Exeter/lab book/gibs/wk3

H2O to 50 µl:

Incubated 36 °C for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Ligation:

Upstream part digest:

Downstream part digest:

Destination plasmid digest:

10X T4 DNA ligase buffer:

T4 DNA ligase:

H2O:

Heat inactivated at 80 °C for 20 minutes

Stored at -20°C prior to afternoon transformation

Standard assembly run in parallel to 3A

Upstream:

DNA (500ng):

SpeI:

10X NEBuffer 2:

100X BSA:

H2O (to 40 µl):

Downstream:

DNA (500ng)

- EcoRI-HF:

XbaI:

10X NEBuffer 2:

100X BSA:

H2O :

Digested for 1 hour at 36 °C

DNA cleanup from enzymatic reactions using microcentrifuge and Quiagen column (n.b. this was intended for gel electrophoresis of upstream fragments to be run in parallel, but the gel was unsuccessful. It later turned out we had the wrong sequences and had not realised the sequences were too short to show on a gel for gel purification.)

For RBS 2 and RBS 4 plasmids (post-digestion)

1.40 µl digest + 120 µl QG buffer + 40 µl isopropanol mixed and transferred to column (n.b. RBS 2 may not have been adequately mixed before being transferred to column)

2.Centrifuged for 1 minute, 13000 rpm, room temp.

3.Flow through discarded

4.500 µl buffer QG added to column

5.Centrifuged for 1 minute, 13000 rpm, room temp.

6.Flow through discarded

7.750 µl buffer PE added to column

8.Centrifuged for 1 minute, 13000 rpm, room temp.

9.Flow through discarded

10.Centrifuged for 1 minute, 13000 rpm, room temp.

11.Moved column to fresh 1.5 ml eppendorf

12.30 µl water added

13.Left to stand for 1 minute

14.Centrifuged 13000 rpm, 1 minute, room temp.

15.DNA stored at -20 °C after analysis with a nanodrop spectrophotometer

Transformation of 3A assembled Pbad strong-RBS and PlacI-RBS

1.2 µl DNA added to 50 µl top10 competent cells

2.Incubated on ice 30 minutes

3.Heat shocked 42 °C, 30 seconds

4.Ice 2 minutes

5.Aseptically added 250 µl S.O.C. medium

6.Shaken 220 rpm, 37 °C, 1 hour

7.Spread plates of 20 µl and 100 µl made on LB(tetracycline) plates

8.Plates incubated at 37 °C overnight

Suspended sequencing primers (according to manufacturer’s instructed volumes) and prepared 50 µl aliquots for later

Resuspended BBa_I0500 (large pbad promoter - selected as alternative to pbad strong due to sequence match with Gibson primers) in 10 µl water

Left to stand for 5 minutes

Transferred to eppendorf

Centrifuged for 5 secs

1.2 µl DNA added to 50 µl top10 competent cells (remaining DNA stored at -20 °C)

2.Incubated on ice 30 minutes

3.Heat shocked 42 °C, 30 seconds

4.Ice 2 minutes

5.Aseptically added 250 µl S.O.C. medium

6.Shaken 220 rpm, 37 °C, 1 hour

7.Spread plates of 20 µl and 100 µl made on LB(kanamycin) plates

8.Plates incubated at room temperature over weekend

Transferred colonies from plates prepared 25.7.12 into 5 ml LB(tetracycline) broth (1:1000)

Incubated overnight at 220 rpm, 37 °C

Miniprep of 3A assembled plasmid (from 25.7.12)

1.Transferred broth to fresh falcon tubes to remove pipette tips (tips and original tubes stored in fridge until same day streak-plates made)

2.Centrifuged 5 minutes, 3901 rcf, 4 °C

3.Supernatant discarded

4.250 µl resuspension solution added

5.Pipetted to mix and transferred to clean 1.5 ml eppendorf

6.250 µl lysis solution added

7.Inverted ~6 X

8.350 µl neutralisation solution added

9.Inverted ~6 X

10.Centrifuged 5 mins, 13000 rpm, room temperature

11.Transferred supernatant to spin column

12.Centrifuged for 1 minute, 13000 rpm, room temperature

13.Discarded flow-through

14.500 µl wash solution added

15.Centrifuged 1 minute, 13000 rpm, room temperature

16.Discarded flow-through

17.500 µl wash solution added

18.Centrifuged 1 minute, 13000 rpm, room temperature

19.Discarded flow-through

20.Centrifuged 1 minute, 13000 rpm, room temperature

21.Transferred column to clean 1.5 ml eppendorf

22.50 µl water added

23.Centrifuged 1 minute, 13000 rpm, room temperature

24.5 µl water added

25.Centrifuged 1 minute, 13000 rpm, room temperature

26.Column discarded, DNA concentration measured and DNA stored in eppendorf at -20 °C

(n.b. digest gel was run for analysis, but for this part of the project is considered irrelevant due to inconclusive results from small fragment size and because BBa-I0500 was decided to be more useful for this part of the project)

Streak plates made from dregs of overnight culture (see above)