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 Pbad W (211.4 ng/µl) Pbad S (130.4 ng/µl) PlacI (299.2 ng/µl) MM DNA (500ng) 2.37 µl 3.83 µl 1.67 µl - EcoRI-HF 0.5 µl 0.5 µl 0.5 µl 2 µl SpeI 0.5 µl 0.5 µl 0.5 µl 2 µl 10X NEBuffer 2 2.5 µl 2.5 µl 2.5 µl 10 µl 100X BSA 0.25 µl 0.25 µl 0.25 µl 1 µl H2O (to 25 µl) 18.88 µl 17.42 µl 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 MM DNA 250ng - EcoRI-HF 0.5 µl 2 PstI 0.5 µl 2 10X NEBuffer 2 2.5 µl 10 100X BSA 0.25 µl 1 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
**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)
**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
Incubated overnight at 220 rpm, 37 °C **Friday 27.7.12** 9 am Miniprep of 3A assembled plasmid (from 25.7.12)
(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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