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Team:UAB-Barcelona/CS1
From 2009.igem.org
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Transformation with biobricks
After designing the biobricks we proceeded to transform those we needed, using a method of chemical transformation and with competent cells prepared with a new improved protocol.
In the first transformation that we did, we had a low efficiency. We only obtained colonies of the biobricks with resistance to kanamycin. Because of it we prepared plates with lower Ampicillin's concentration (50 g/ml).
We increased the efficiency of transformation adding one more step to our protocol: 1 min in ice after the 45 s in 42ºC.
We extracted the plasmidic DNA (minipreps) with the kit Wizard Plus SV Minipreps DNA Purification System (Promega) (PROTOCOL).
Cloning strategy
1st roundphoa (E,S) + backbone BBa_K093005 (E,S) // Biobrick 1 nirk (E,S) + backbone BBa_K093005 (E,S) // Biobrick 2 nsr (X,P) + backbone BBa_K093005 (E,S) // Biobrick 3
pLacI-RBS-RFP-T-T BBa_J04450 (E,S) + linker BBa_B0040 (E,X) // Biobrick 5 ptet-RBS-RFP-T-T BBa_I13521 (E,S) + linker BBa_B0040 (E,X) // Biobrick 6
RBS BBa_J61127 (S,P) + nsr (X,P) // Biobrick 8 ptet BBa_R0040 (S,P) + Biobrick 8 (X,P) // Biobrick 9 placI BBa_K091112 (S,P) + Biobrick 8 (X,P) // Biobrick 10
2nd roundBiobrick 4 (X,P) + RBS-GFP-T-T BBa_I13504 (S,P) // Biobrick 11 Biobrick 7 (X,P) + RBS-RFP-T-T BBa_J04450 (S,P) // Biobrick 12 3rd roundBiobrick 11 + Biobrick 5 // Biobrick 13 Biobrick 11 + Biobrick 6 // Biobrick 14 Biobrick 9 (X,P) + RBS-GFP-T-T BBa_I13504 (S,P) // Biobrick 15 Biobrick 10 (X,P) + RBS-GFP-T-T BBa_I13504 (S,P) // Biobrick 16 4th roundBiobrick 12 + Biobrick 15 // Biobrick 17 Biobrick 12 + Biobrick 16 // Biobrick 18
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Biobrick digestion
Agarose gel digestion purification.
From left side to right: BBa_J04450 digested and no digested; BBa_I13521 digested y no digested; lambda-DNA hinIII; nirk ; nsr; phoa
Agarose gel digestion purification.
From left side to right: BBa_B0014 digestedand no digested; lambda-DNA hinIII; BBa_J61127 digested and no digested; lambda-DNA hinIII ; nirk ; BBa_B0040 digested and no digested.
Agarose gel digestion purification.
From left side to right: BBa_E0840 digested and no digested; lambda-DNA hinIII; BBa_I13504 digested and no digested; lambda-DNA hinIII; nirk ; BBa_K093005 digested and no digested.
