SCU Weekly

 

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6.8~6.15 1. Transformation of BBa_B0015, BBa_CO261 to psb1AK3 is successful 2. Transformed pSB1T3, pSB1K3, pSB1C3, pSB1A3, BBa_I13504 and BBa_I13504. 4. The bacteria precipitate transformed with pSB1C3 and pSB1A3 were red after centrifugation, the degree of red decreased in this order: pSB1K32 (red) - pSB1A3 (pink) - pSB1A3(tinge) - pSB1K3(yellow). 5. Chloramphenicol was insoluble，and it was tested to be useless using DH5α. 6. Prepare A + , K + ,T + ,C + plates and antibiotic store liquor. 7. Digested and assembled BBa_CO261, pSB1C3, then transformed them 9. Digestion of BBa_I13504 and BBa_B0015, and results proved that the transformation was successful 6.16~6.22 1. PCR to amplify 8B but failed. 2. Chloramphenicol was tested to be effective 3. Transformed pSB1T3, BBa_CO261, BBa_B0015, BBa_I13504, pSB1K3 (pSB1K3) and pSB1A3(pSB1A3) 4. Plasmid extraction of BBa_F2622 + BBa_CO261 5. Digested BBa_F2622 + BBa_CO261 and pSB1T3 ( EcoRⅠand PstⅠ) 6. Assembled BBa_F2622 + BBa_CO261, BBa_B0015 and pSB1T3 6.23~6.29 1. Strain conservation of BBa_F2622 + BBa_CO261, pSB1C3 2. Prepared ddH2O 3. Added 1µl Dpn Ӏ into pSB1T3 (pSB1T3) reaction system when digesting 6.30~7.6 1. Digested BBa_F2622 + BBa_CO261 and pSB1T3 ( EcoRⅠand PstⅠ) 2. Assembled BBa_F2622 + BBa_CO261, BBa_I13504, and pSB1T3, but failed to transform them 7.7~7.13 1. Plated BBA_F2622 + BBa_CO261 + BBa_I13504 (1.2.3) and BBA_F2622 + BBa_CO261 + BBa_B0015 (1.2.3) which were assembled last week but failed. 2. Cultured BBa_B0015 1 and 2 7.14~7.20 1. No much experiment 7.21~7.27 1. Failed to amplify pSB1K3, pSB1A3, pSB1C3 through PCR 2. Transformed BBa_I61046 (RBS + Cre), K5-R0040 and they were successful 3. Extracted plasmid of pSB1T3, BBA_F2622 + BBa_CO261 and only pSB1T3was successful 4. Incubated BBa_R0040, BBa_F2622 + BBa_CO261 + BBa_I13504, BBA_F2622 + BBa_CO261 + BBa_B0015 5. Successfully assembled BBA_F2622 + BBa_CO261 + BBa_I13504, BBA_F2622 + BBa_CO261 + BBa_B0015 RePCR by taq enzyme, we got experiences: Taq is suitable for amplifying snippets of 2kb in size, and the reaction is fast 10X taq buffer with (NH4)2SO4 reduces nonspecification 2mM Mg2 + is suitable for taq Avoid DNA pollution during template preparation No pollution of raw material, it would be better to operate on the laminar Negative control is necessary. 7.28~8.2 1. Recovered PCR product of 27th 2. Assembled BBa_F2622 + BBa_CO261 + BBa_I13504, BBa_F2622 + BBa_CO261 + BBa_B0015 with pSB1C3 carrier, then did transformation and incubate 3. Incubated R0040, pSB1T3, BBa_R0062, BBa_F2622 + BBa_CO261, BBa_B0015 4. Replated BBa_F2622 + BBa_CO261 + BBa_I13504, BBa_F2622 + BBa_CO261 + BBa_B0015 5. Digested BBa_R0040, BBa_R0062, BBa_J61064 6. Assembled BBa_R0062with BBa_J61064, then did transformation 8.3~8.10 1. Incubated BBa_R0062+BBa_J61064, and gel analysis of plasmid extraction and digestion products proved that the ligation was successful 2. Transformed BBa_I746361, BBa_K145215, BBa_K145215. 3. Digested BBa_R0062, BBa_J61064, pSB1C3, and assembled them overnight 8.11~8.17 1. Transformed, BBa_I746361, BBa_K145215，BBa_J23032，BBa_F2622 + BBa_CO261 2. Extracted plasmid of BBa_K145215, BBa_F2622 + BBa_CO261 3. Digested BBa_K145215, BBa_CO261, BBa_F2622 + BBa_CO261， 4. Incubated BBa_F2622，BBa_J23032 5. Sent BBa_K145215, BBA_F2622 to biotech company to sequence them. 8.18~8.24 1. Assembled BBa_R0040 + BBa_J23032 (Ptet + lock), PO + LOX + BBa_R0040 2. Transformed BBa_R0062 + BBa_I718008, BBa_F2622 + BBa_CO261, BBa_R0040 + BBa_J23032 3. Incubated BBa_R0040 + BBa_J23032, BBa_R0062 + BBa_I718008 4. Extracted plasmid of BBa_J23032, B0034（strong RBS, BBa_R0040 + BBa_J23032, BBa_R0062 + Cre and then did digestion. 5. Digested BBa_I718008, BBa_R0062 + BBa_I718008, BBa_B0034. 6. Sent BBa_R0062 + BBa_I718008to sequence, however all turned out to be wrong, so we need to reassembly them. 7. Sent BBa_R0040 + BBa_J23032, to sequence 8. Amplified BBa_I746361 through PCR 8.25~8.31 1. Digested BBa_R0040 + BBa_J23032 and BBa_F2622 + BBa_B0015, BBa_R0062 + BBa_I718008 2. Designed primer of APO 3. Failed to PCR Cre + ssrA 4. Transformed BBa_R0040 + PO, 5. Recultured BBa_R0062 + BBa_I718008, BBA_F2622 + BBa_CO261 6. Single digested BBA_F2622 + BBa_B0015,R0040 + Po, compared with previous single digested BBa_B0015, but failed. 7. Redigested BBa_B0015, then did ligation and transformation. 8. Sent to sequence: BBa_R0040 + BBa_J23032, BBa_R0062 + BBa_I718008, R0040 + Po, 9. Recovered BBa_B0015 and BBa_R0040 from conservation 9.1~9.7 1. succeeded in amplifying Cre + ssrA by PCR 2. Digested BBa_R0040, pSB1C3, pSB1K3, pSB1A3, pSB1T3, BBa_R0062 + BBa_I718008, BBA_F2622 + BBa_CO261, Cre + BBa_B0015, PCRed BBA_F2622 + BBa_B0015, BBA_F2622 + BBa_B0015 + R0040 + J23032 3.Assembled BBa_I718008 + BBa_B0015, BBa_F2622 + BBa_CO261(pSB1C3), R0040 + J23032 + BBA_F2622 + BBa_B0015(pSB1T3), BBa_R0040 + po + BBa_I13507 (pSB1T3), RBS + BBa_I718008 + ssrA (pSB1C3) ,BBa_F2622 + BBa_B0015 (pSB1C3) BBa_R0040 + BBa_I13504 (pSB1C3), BBa_I746361 + BBa_I13504 (pSB1C3), BBa_R0040 → BBa_B0015 (pSB1C3), then transformed them. 4. Cultured BBa_R0062 + BBa_I718008 5. Digested pSB1C3, pSB1K3, pSB1A3, pSB1T3, BBa_R0062 + BBa_I718008, BBa_F2622 + BBa_CO261 6. Transformed Cre + BBa_B0015, pSB1A2, BBa_J04650 7. Transformed pSB1A2, BBa_J04650 8. Sent to sequence: BBa_R0062 + BBa_I718008, BBa_F2622 + BBa_B0015 → 1B0034 9. Did gel extraction RBS + Cre + ssrA 9.8~9.14 1. Digested BBa_I13507, pSB1C3, BBa_R0040 + Po, BBa_R0040 + po + BBa_I13507 2. Incubated BBa_R0040→BBa_B0015 3. Assembled BBa_R0040 + po + BBa_I13507, BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + po + BBa_I13507 4. Sent to sequence: BBa_B0034→BBa_B0015, BBa_R0040→BBa_B0015, BBa_R0040 + PO + BBa_I13507 5.96-well plates experiment of BBa_R0040 + PO 6. Cultured BBa_R0040 + BBa_I13504 and observed its fluorescence 7. Transformed BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + Po + BBa_I13507 9.15~9.21 1. Miniprep: BBa_I746361 + BBa_I13507, BBa_R0040 + BBa_ J23032 + BBa_J04650, BBa_R0040 + Po + BBa_I13507, BBa_I746361 + BBa_I13507, BBa_I746361 + BBa_I13507 + BBa_R0040→BBa_B0015, BBa_K145215 + BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_I746361 + BBa_I13507 + BBa_R0040 + BBa_J23032 + BBA_F2622 + BBa_B0015 + BBa_K145215, BBa_R0040 + Po + BBa_I13507, BBa_I746361→BBa_B0015 + BBa_K145215, BBa_I13521, BBa_K145215 + BBa_R0040 + BBa_J23032 + BBa_J04650 2. Digestion: BBa_I746361 + BBa_I13507, BBa_I746361 + BBa_I13507 + BBa_R0040→BBa_B0015, BBa_K145215 + BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + Po + BBa_I13507, BBa_R0040 + Po + BBa_I13507, BBa_I746361→BBa_B0015 + BBa_K145215 3. Incubate: BBa_I746361 + BBa_I13507, BBa_I746361 + BBa_I13507 + BBa_R0040→BBa_B0015, BBa_I718008 + Po + BBa_I13507, BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_K145215 + BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040, BBa_K145215 + BBa_B0034 + BBa_J23032 + BBa_B0015, R0040 + Po + BBa_I13507 4. Prepared A + and T + plates 5. Assembled BBa_I746361→BBa_B0015 + BBa_K145215 and BBa_R0040 + BBa_I13507, then did transformation 6. Function test: BBa_K145215 + BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + BBa_J23032 + BBa_J04650 + BBa_K145215, BBa_I13521, BBa_I746361 + BBa_I13507, BBa_I746361 + BBa_I13507 + BBa_R0040→BBa_B0015, BBa_R0040 + Po + BBa_I13507 7. Gel extraction of BBa_R0040 + Po + BBa_I13507 8. Sent BBa_R0040 + BBa_J23032 + BBa_J04650 + BBa_K145215, BBa_I746361 + BBa_I13507 + BBa_R0040→BBa_B0015 to sequence 9.22-9.28 1. Prepare LB incubate and LB plates 2. Incubated BBa_B0034→BBa_B0015, and then extracted plasmid 4. Digested BBa_I13521, BBa_I718008, BBa_B0034→BBa_B00155. Gel extraction of BBa_I718008, and BBa_I13507 6. Function test of BBa_I13521, BBa_I746361→BBa_B0015, BBa_I746361→ BBa_K145215, BBa_I746361 + BBa_I13507 7. PCR BBa_I13507 8. Assembled BBa_I718008 and BBa_B0034→BBa_B0015 |} __NOEDITSECTION__ __NOTOC__