Team:Exeter/lab book/gibs/wk3

Operon Construction  |  Glycobase

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Mary, Freddie, Becca and Alex B.

9am

3A assembly of Pbad weak-RBS, Pbad strong-RBS and PlacI-RBS

Upstream

For 500ng DNA:

Pbad Weak - (211.4 ng/µl)=2.37 µl

Pbad Strong -(130.4 ng/µl) =3.83 µl

PlacI - (299.2 ng/µl)=1.67 µl

EcoRI-HF: 0.5 µl (master mix volume 2 µl)

SpeI: 0.5 µl (master mix volume 2 µl)

10X NEBuffer 2: 2.5 µl (master mix volume 10 µl)

100X BSA: 0.25 µl (master mix volume 1 µl)

H2O (to 25 µl):

Pbad Weak =18.88 µl

Pbad Strong =17.42 µl

PlacI =19.58 µl

Downstream

For 500 ng DNA:

RBS (77.4 ng/µl)=6.46 µl

XbaI: 0.5 µl

PstI: 0.5 µl

10X NEBuffer 2: 2.5 µl

100X BSA: 0.25 µl

H2O to 25 µl: 14.79 µl

Destination plasmid

DNA 250ng

EcoRI-HF: 0.5 µl (master mix vol. 2 µl)

PstI: 0.5 µl (master mix vol. 2 µl)

10X NEBuffer 2: 2.5 µl (master mix vol. 10 µl)

100X BSA: 0.25 µl (master mix vol. 1 µl)

H2O to 25 µl

Incubated 36 °C for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Ligation:

Upstream part digest: 2 µl

Downstream part digest: 2 µl

Destination plasmid digest: 2 µl

10X T4 DNA ligase buffer: 2 µl

T4 DNA ligase: 1 µl

H2O: 11 µl

Incubated at room temperature for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Stored at -20°C prior to afternoon transformation

2.45pm

Resuspended DNA according to IDT resuspension protocol (Alex B. and Becca then transformed them)

1.2 µl of DNA from Pbad S-RBS and PlacI-RBS ligations added to 25 µl top10 competent cells

2.Incubated on ice for 30 minutes

3.Heat shocked 42 °C 30 seconds

4.Placed on ice for 2 minutes

5.Aseptically added 250 µl S.O.C. medium (pre-warmed)

6.Shaken at 220 rpm, 37 °C, 1 hour

7.Spread plates of 20 and 100 µl made

8.incubated at 37 °C overnight

Mary and Freddie

9.45 am

Biobricking attempt 2

To make Pbad Strong-RBS and PlacI-RBS (n.b. placI-RBS relevant only to 3 gene part of project)

3A assembly

Upstream Pbad S (130.4 ng/µl) PlacI (299.2 ng/µl) MM DNA (500ng) 3.8 µl 1.7 µl - EcoRI-HF 1 µl 1 µl 3 µl SpeI 1 µl 1 µl 3 µl 10X NEBuffer 2 5 µl 5 µl 15 µl 100X BSA 0.5 µl 0.5 µl 1.5 µl H2O (to 50 µl) 38.7 µl 40.8 µl - Downstream RBS (77.4 ng/µl) MM DNA (500ng) 6.5 µl - XbaI 0.5 µl 3 PstI 0.5 µl 3 10X NEBuffer 2 2.5 µl 15 100X BSA 0.25 µl 1.5 H2O 6.7 µl - Destination plasmid (tetracycline) pSB1T3 (linear) MM DNA 250ng 20 µl - EcoRI-HF 1 µl 3 PstI 1 µl 3 10X NEBuffer 2 5 µl 15 100X BSA 0.5 µl 1.5 H2O to 50 µl 22.5 µl - Incubated 36 °C for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Ligation: Upstream part digest 2 µl Downstream part digest 2 µl Destination plasmid digest 2 µl 10X T4 DNA ligase buffer 2 µl T4 DNA ligase 1 µl H2O 11 µl Incubated at room temperature for 10 minutes

Heat inactivated at 80 °C for 20 minutes

Stored at -20°C prior to afternoon transformation

Standard assembly run in parallel to 3A

Upstream Pbad S PlacI MM DNA (500ng) 15.3 µl 6.7 µl - EcoRI-HF 1 µl 1 µl 3 µl SpeI 1 µl 1 µl 3 µl 10X NEBuffer 2 5 µl 5 µl 15 µl 100X BSA 0.5 µl 0.5 µl 1.5 µl H2O (to 40 µl) 17.2 µl 25.8 µl - Downstream RBS 2 (77.4 ng/µl) RBS 4 (63.0 ng/µl) MM DNA (500ng) 25.8 µl 31.7 µl - EcoRI-HF 1 µl 1 µl 3 XbaI 1 µl 1 µl 3 10X NEBuffer 2 5 µl 5 µl 15 100X BSA 0.5 µl 0. 5 µl 1.5 H2O 6.7 µl 0.8 - Digested for 1 hour at 36 °C

DNA cleanup from enzymatic reactions using microcentrifuge and Quiagen column

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

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)