Team:Exeter/lab book/gibs/wk3
From 2012.igem.org
Operon Construction: 23rd - 27th July 2012 **Tuesday 24.7.12**Mary, Freddie, Becca and Alex B. 9am 3A assembly of Pbad weak-RBS, Pbad strong-RBS and PlacI-RBS Upstream For 500ng DNA:
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 Strong =17.42 µl PlacI =19.58 µl Downstream RBS (77.4 ng/µl) DNA (500ng): 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
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 **Wednesday 25.7.12** 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)
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
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 **Thursday 26.7.12** 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
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
Incubated overnight at 220 rpm, 37 °C **Friday 27.7.12** 9 am Miniprep of 3A assembled plasmid (from 25.7.12)
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)
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