Team:Shenzhen SZMS/Lab

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[[Team:Shenzhen SZMS|<<Back to main page]]
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{| class="wikitable" align=center
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=Schedule=
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|align=center width=12.5%|[[Team:Shenzhen SZMS|Home]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS/Team Members|Team Members]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS/Project|Project]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS/Modelling|Modelling]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS/Lab|Lab]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS/iGem Safety Q&A|Safety]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS/Gallery|Gallery]]
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|align=center width=12.5%|[[Team:Shenzhen SZMS#Human Practice|Human Practice]]
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|}
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[https://2014hs.igem.org/Main_Page<br> <<Back to wiki main page]
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----
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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]].
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=Log Book=
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==Schedule==
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{| class="wikitable"
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|-
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!width=50px|No. !!width=300px| Procedure !!width=100px|Backbone !!width=250px| Status of the Plasmid
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|-
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|1
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|[K115016] Spe1,Pst1 Double Digestion, Electrophoresis detection，DNA refinement;[C0051] X ba1,Pst1Double Digestion, Gel extraction;Combination
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|1C3(K115016)
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|[K115016+C0051]
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|-
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|2
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|[J23106] Spe1,Pst1 Double Digestion, Electrophoresis detection，DNA refinement;[K115016+C0051] X ba1,Pst1Double Digestion, Gel extraction;Combination
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|1A2(J23106)
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|J23106+[K115016+C0051]
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|-
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|3
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|[J2s3106+ K115016+C0051] EcoR1,Spe1Double Digestion, Electrophoresis detection，DNA refinement;[B0051] EcoR1,Xba1 Electrophoresis detection，DNA refinement;Combination
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|1C3(B0015)
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|[J23106+K115016+C0051]+B0015
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|-
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|4
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|[B0030] Spe1,Pst1 Double Digestion, Electrophoresis detection, DNA refinement;[EC:1.7.7.2] Xba1, Pst1Double Digestion, Gel extraction;Combination
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|1A2(B0030)
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|B0030+EC:1.7.7.2
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|-
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|5
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|[R0051] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[B0030+ EC:1.7.7.2] Xba1,Pst1 Double Digestion, Gel extraction;Combination
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|1A2(R0051)
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|R0051+[B0030+ EC:1.7.7.2]
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|-
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|6
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|[B0030] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[K145015] Xba1,Pst1Double Digestion, Gel extraction; Combination
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|1A2(B0030)
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|B0030+K145015
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|-
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|7
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|[B0030+ K145015] EcoR1, Spe1Double Digestion, Electrophoresis detection,DNA refinement;[B0015] EcoR1, Xba1Double Digestion,Electrophoresis detection,DNA refinement;Combination
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|1C3(B0015)
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|[B0030+ K145015]+B0015
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|-
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|8
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|[R0051+B0030+ EC:1.7.7.2] Spe1, Pst1Double Digestion,Gel extraction;[B0030+ K145015+B0015]Xba1, Pst1 Gel extraction;Combination
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|1C3(B0030+K145015+B0015)
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|[R0051+B0030+ EC:1.7.7.2]+[B0030+K145015+B0015]
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|-
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|9
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|[K115017] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[C0051] Xba1, Pst1Double Digestion,Gel extraction; Combination
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|1C3(K115016)
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|[K115017+C0051]
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|-
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|10
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|[J23106] Spe1,Pst1Double Digestion, Electrophoresis detection,DNA refinement;[K115017+C0051] Xba1, Pst1Double Digestion, Gel extraction;Combination
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|1A2(J23106)
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|J23106+[K115017+C0051]
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|-
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|11
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|[J23106+ K115017+C0051] EcoR1, Spe1Double Digestion,Gel extraction;[B0015]EcoR, Xba1Double Digestion, Electrophoresis detection,DNA refinement,Combination
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|1C3(B0015)
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|[J23106+ K115017+C0051]+B0015
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|-
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|12
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|[The first part] Spe1,Pst1 Double Digestion;[The second part] Xba1,PstL Double Digestion;Gel extraction; Combination
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|1C3(The first part)
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|1st+2nd
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|-
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|13
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|[The first and second parts] Spe1、Pst1Double Digestion; [The third part] Xba1、Pst1Double Digestion;Gel extraction; Combination
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|1C3(The third part)
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|1st +2nd+3rd<br>Done
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|}
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==Log Book==
{| class="wikitable"
{| class="wikitable"
|-
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Latest revision as of 03:56, 21 June 2014

Home Team Members Project Modelling Lab Safety Gallery Human Practice


<<Back to wiki main page

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.
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