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Team:Shenzhen SZMS/Lab
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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. |
