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Here you can find out how we build our plasmid. The plasmid map and information of parts used is given in Team:Shenzhen SZMS/Project.

Schedule

No.	Procedure	Backbone	Status of the Plasmid
1	[K115016] Spe1,Pst1 Double Digestion, Electrophoresis detection，DNA refinement;[C0051] X ba1,Pst1Double Digestion, Gel extraction;Combination	1C3(K115016)	[K115016+C0051]
2	[J23106] Spe1,Pst1 Double Digestion, Electrophoresis detection，DNA refinement;[K115016+C0051] X ba1,Pst1Double Digestion, Gel extraction;Combination	1A2(J23106)	J23106+[K115016+C0051]
3	[J2s3106+ K115016+C0051] EcoR1,Spe1Double Digestion, Electrophoresis detection，DNA refinement;[B0051] EcoR1,Xba1 Electrophoresis detection，DNA refinement;Combination	1C3(B0015)	[J23106+K115016+C0051]+B0015
4	[B0030] Spe1,Pst1 Double Digestion, Electrophoresis detection, DNA refinement;[EC:1.7.7.2] Xba1, Pst1Double Digestion, Gel extraction;Combination	1A2(B0030)	B0030+EC:1.7.7.2
5	[R0051] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[B0030+ EC:1.7.7.2] Xba1,Pst1 Double Digestion, Gel extraction;Combination	1A2(R0051)	R0051+[B0030+ EC:1.7.7.2]
6	[B0030] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[K145015] Xba1,Pst1Double Digestion, Gel extraction; Combination	1A2(B0030)	B0030+K145015
7	[B0030+ K145015] EcoR1, Spe1Double Digestion, Electrophoresis detection,DNA refinement;[B0015] EcoR1, Xba1Double Digestion,Electrophoresis detection,DNA refinement;Combination	1C3(B0015)	[B0030+ K145015]+B0015
8	[R0051+B0030+ EC:1.7.7.2] Spe1, Pst1Double Digestion,Gel extraction;[B0030+ K145015+B0015]Xba1, Pst1 Gel extraction;Combination	1C3(B0030+K145015+B0015)	[R0051+B0030+ EC:1.7.7.2]+[B0030+K145015+B0015]
9	[K115017] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[C0051] Xba1, Pst1Double Digestion,Gel extraction; Combination	1C3(K115016)	[K115017+C0051]
10	[J23106] Spe1,Pst1Double Digestion, Electrophoresis detection,DNA refinement;[K115017+C0051] Xba1, Pst1Double Digestion, Gel extraction;Combination	1A2(J23106)	J23106+[K115017+C0051]
11	[J23106+ K115017+C0051] EcoR1, Spe1Double Digestion,Gel extraction;[B0015]EcoR, Xba1Double Digestion, Electrophoresis detection,DNA refinement,Combination	1C3(B0015)	[J23106+ K115017+C0051]+B0015
12	[The first part] Spe1,Pst1 Double Digestion;[The second part] Xba1,PstL Double Digestion;Gel extraction; Combination	1C3(The first part)	1st+2nd
13	[The first and second parts] Spe1、Pst1Double Digestion; [The third part] Xba1、Pst1Double Digestion;Gel extraction; Combination	1C3(The third part)	1st +2nd+3rd Done

Log Book

Date	Event	Results	Problem summary
Apr.3	B0015(chl) B0030(chl) Plasmid Transformation	Done (yet this B0030 is single error)
Apr.4	Single clone selected from the work on Apr.3 (In order to be pithy, this process is omitted from the table in all the following daily work)	Done
Apr.5	Plasmid preped and culture restored from the bacterial suspension on Apr.4 (omitted later for pithiness and convenience as well)	Done
Apr.6	K115016(CHL) K115017(CHL) C0051(CHL) transformation	Done
May.2	J23106(AMP) K082003(CHL，as GFP+LVA) transformation (help offered by Mentor Wang)	Done
May.5	Digestion: K115016(S/P) C0051(X/P) C0051(E/S) B0015(E/X) Electrophoresis Gel extraction 16℃ overnight	Done
May.6	Product from 5.5（CHL）transformed	Failed	Might be the problem during the plasmid prep
May.7	Plasmid mentioned above repreped	Done
May.13	Digestion: C0051(E/S) B0015(S/P) K082003(E/S) Electrophoresis Gel extraction 16℃ overnight	Done, C0051-B0015 K082003-B0015 gained
May.14	Products from 5.13（CHL）all transformed	Done, C0051-B0015 K082003-B0015 gained
May.15	Gel confirmation (products from 5.14), comparison with gel of B0015	Done, product longer than the original plasmid
May.27	Digestion: C0051-B0015(X/P) K115016(S/P) K115017(S/P) Electrophoresis Gel extraction Combination Transformation（CHL）	Done, K115016-C0051-B0015 K115017-C0051-B0015 gained.
May.28	B0030(AMP) transformation	Done; informed that R0051 requires PCR in order to get the primer of EC 1.7.7.2
May.29	Digestion: J23106(S/P) K115016-C0051-B0015(X/P) K115017-C0051-B0015(X/P) Electrophoresis Gel extraction Combination Transformation（AMP）	Done, J23106-K115016-C0051-B0015(27℃CI-protein) J23106-K115017-C0051-B0015（32℃CI-protein） gained.
May.30	The result of sequencing of C0051-B0015 K082003-B0015 received	Correctly combined
Jun.2	Digestion: B0030(S/P) K082003-B0015(X/P) Electrophoresis Gel extraction Combination Transformation（AMP）	Done
Jun.8	The results of sequencing of J23106-K115016-C0051-B0015(27℃CI) and J23106-K115017-C0051-B0015（32℃ CI）received.	Correctly combined
Jun.9	Digestion:B0030-K082003-B0015(E/S) J23106-K115017-C0051-B0015(E/X) Electrophoresis Gel extraction Combination Transformation（AMP）	Done with transformation; Yet failed in the electrophoretic analysis.	Disconnected
Jun.10		ciF1 ciF2 ciF3: primers to synthesize EC 1.7.7.2 and PEP (additional PEP R0051-B0030-) gained.
Jun.11	With the original blue 2X Master Mix PCR round 1,2,3 implemented	Failed; only primer founded in the electrophoretic analysis
Jun.12	Round 1 from 6.11 repeated in the morning; Round 3 PCR with 2X hi-fi Master Mix at noon	Failed in the morning but done in the afternoon; R0051-B0030-1.7.7.2 recycled product gained.	might be the problem of MIX
Jun.13	Digestion: 27℃ CI（S/P）R0051-B0030-1.7.7.2（X/P） 32℃ CI（E/X） B0030-K082003-B0015(E/S) Electrophoresis Gel extraction Combination Transformation.	Failed, no culture founded on the dish.	Unknown error; might be the problem of extracted gel.
Jun.15	Digestion: 27℃ CI（S/P）R0051-B0030-1.7.7.2（X/P） 32℃ CI（E/X） B0030-K082003-B0015(E/S) Electrophoresis Gel extraction Combination Transformation.	According to the result of electrophoresis, the length of the digested and combined part is the same as that of the primitive plasmid; yet we cannot exclude the problem of plasmid prep; might have failed.	A tube was missed during the digestion yet was made up later; do not know about its possible effect yet.