SCU Weekly

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==June==
 +
===6.8~6.15===
 +
*transformation of BBa_B0015，BBa_CO261 to psb1AK3 is successful
 +
*Transformed pSB1T3,pSB1K3, pSB1C3,pSB1A3, BBa_I13504 and BBa_I13504.
 +
*the bacteria precipitate transformed with pSB1C3 and pSB1A3  were red after centrifugation, the degree of red decreased in this order: pSB1K32 (pinkred) - pSB1A3 (pink) - pSB1A3(tinge) - pSB1K3(yellow).
 +
*Chloramphenicol was insoluble，and it was tested to be useless using DH5α.
 +
*prepare A + ,K + ,T + ,C +  plates and antibiotic store liquor.
 +
*Digested and ligated  BBa_CO261, pSB1C3,then transformed them
 +
*Digestion of  BBa_I13504, BBa_B0015, and proved that the transformation was successful
 +
===6.16~6.22===
 +
*1.PCR to amplified 8B but failed
 +
*2. Chloramphenicol was tested to be effective
 +
*3.transformed pSB1T3, K2-8B BBa_CO261, BBa_B0015, BBa_I13504, pSB1K3(pSB1K3) and pSB1A3(pSB1A3)
 +
*4.plasmid extraction of  F2622 + BBa_CO261
 +
*5.Digested F2622 + BBa_CO261 and pSB1T3 (EcoRⅠand PstⅠ)
 +
*6.ligated F2622 + BBa_CO261, BBa_B0015 and pSB1T3
-
6.8~6.15
+
===6.23~6.29===
-
1. Transformation of BBa_B0015, BBa_CO261 to psb1AK3 is successful
+
*1.strain conservation of F2622 + BBa_CO261, pSB1C3
-
2. Transformed pSB1T3, pSB1K3, pSB1C3, pSB1A3, BBa_I13504 and BBa_I13504.
+
*2.prepared ddH2O
-
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).
+
*3.Added 1µl Dpn Ӏ into pSB1T3(pSB1T3) reaction system when digesting
-
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
+
==July==
-
1. PCR to amplify 8B but failed.
+
===6.30~7.6===
-
2. Chloramphenicol was tested to be effective
+
*1.Digested F2622 + BBa_CO261 and pSB1T3(EcoRⅠand PstⅠ)  
-
3. Transformed pSB1T3, BBa_CO261, BBa_B0015, BBa_I13504, pSB1K3 (pSB1K3) and pSB1A3(pSB1A3)
+
*2.ligated F2622 + BBa_CO261, BBa_I13504, and pSB1T3, but failed to transform
-
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
+
===7.7~7.13===
-
1. Strain conservation of BBa_F2622 + BBa_CO261, pSB1C3
+
*1.plated F2622 + BBa_CO261 + BBa_I13504(1.2.3) and F2622 + BBa_CO261 + BBa_B0015(1.2.3) which were ligated  last week but failed.
-
2. Prepared ddH2O
+
*2.cultured BBa_B0015 1 and 2
-
3. Added 1µl Dpn Ӏ into pSB1T3 (pSB1T3) reaction system when digesting
+
-
6.30~7.6
+
===7.14~7.20===
-
1. Digested BBa_F2622 + BBa_CO261 and pSB1T3 ( EcoRⅠand PstⅠ)
+
1.no much experiment
-
2. Assembled BBa_F2622 + BBa_CO261, BBa_I13504, and pSB1T3, but failed to transform them
+
-
7.7~7.13
+
===7.21~7.27===
-
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.
+
*1. failed to amplify pSB1K3, pSB1A3, pSB1C3 through PCR
-
2. Cultured BBa_B0015 1 and 2
+
*2. transformed K2-3G(RBS + Cre), K5-24B, K5-R0040 and 3G, 24B were successful
-
 
+
*3.extracted plasmid of K2-8B, F2622 + BBa_CO261 and only K2-8B was successful  
-
7.14~7.20
+
*4.Liquid cultured 24B, 3G, R0040, 6O, 7F, 12C and F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015, 4F.
-
1. No much experiment
+
*5. Successfully ligated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015
-
 
+
-
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:
RePCR by taq enzyme, we got experiences:
Taq is suitable for amplifying snippets of 2kb in size, and the reaction is fast
Taq is suitable for amplifying snippets of 2kb in size, and the reaction is fast
-
10X taq buffer with (NH4)2SO4 reduces nonspecification
+
10X taq buffer with (NH4)2SO4 reduces nonspecification,
-
2mM Mg2 +  is suitable for taq
+
B0034M Mg2 +  is suitable for taq
Avoid DNA pollution during template preparation
Avoid DNA pollution during template preparation
No pollution of raw material, it would be better to operate on the laminar
No pollution of raw material, it would be better to operate on the laminar
Negative control is necessary.
Negative control is necessary.
-
7.28~8.2
+
===7.28~8.2===
-
1. Recovered  PCR product of 27th
+
*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
+
*2.ligated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015 with pSB1C3 carrier, then did transformation and liquid culture
-
3. Incubated R0040, pSB1T3, BBa_R0062, BBa_F2622 + BBa_CO261, BBa_B0015
+
*3.Liquid cultured R0040, 8B, 6O, F2622 + BBa_CO261, BBa_B0015
-
4. Replated BBa_F2622 + BBa_CO261 + BBa_I13504, BBa_F2622 + BBa_CO261 + BBa_B0015
+
*4.Replated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015
-
5. Digested BBa_R0040, BBa_R0062, BBa_J61064
+
*5.Digested R0040, 6O, 24B
-
6. Assembled BBa_R0062with BBa_J61064, then did transformation
+
*6.Ligated 6O with 24B, then did transformation
-
 
+
*7.transformed 18O, 19C
-
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
+
== August==
-
1. Assembled BBa_R0040 + BBa_J23032 (Ptet + lock), PO + LOX + BBa_R0040
+
===8.3~8.10===
-
2. Transformed BBa_R0062 + BBa_I718008, BBa_F2622 + BBa_CO261, BBa_R0040 + BBa_J23032
+
*1.ligated 6N + 19C + BBa_B0015, after failing to ligate 6N(T7, promoter) with 19C(reporter)
-
3. Incubated BBa_R0040 + BBa_J23032, BBa_R0062 + BBa_I718008
+
*2.Liquid cultured 6O + 24B, and gel analysis of plasmid extraction and digestion products proved that the ligation was successful
-
4. Extracted plasmid of BBa_J23032, B0034（strong RBS, BBa_R0040 + BBa_J23032, BBa_R0062 + Cre and then did digestion.
+
*3.Transformed K3- I746361, K3-K145215, K2-15J and 5J + 2C. K145215 and 15J turned out to be a failure. Liquid cultured the rest.
-
5. Digested BBa_I718008, BBa_R0062 + BBa_I718008, BBa_B0034.
+
*4.Digested 6O, 24B, pSB1C3, and ligated them overnight
-
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
+
===8.11~8.17===
-
1. Digested BBa_R0040 + BBa_J23032 and BBa_F2622 + BBa_B0015, BBa_R0062 + BBa_I718008
+
*1.transformed 5J + 2C，18O, 19C， I746361,K145215,15J，K5-J23032，F2622 + BBa_CO261
-
2. Designed primer of APO
+
*2.Extracted plasmid of K145215,15J,5J + 2C,F2622 + BBa_CO261
-
3. Failed to PCR Cre + ssrA
+
*3.Digested K145215，6N + I13507，BBa_CO261，5J + 2C,F2622 + BBa_CO261，
-
4. Transformed BBa_R0040 + PO,  
+
*4.Liquid cultured F2622，6N + 19C + BBa_B0015，J23032
-
5. Recultured BBa_R0062 + BBa_I718008, BBA_F2622 + BBa_CO261
+
*5.Sent K145215, 6N + I13507, F2622, 6N + 19C(PSN1C3), 6N + 19C + BBa_B0015(PSB1C3) to biotech company to sequence them.
-
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
+
===8.18~8.24===
-
1. succeeded in amplifying Cre + ssrA by PCR
+
*1.Ligated R0040 + J23032(Ptet + lock) ,B0034 + 2C , PO + LOX + R0040
-
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
+
*2.Transformed B0034 + 6N,6O + 24B,F2622 + BBa_CO261, 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.
+
*3.Liquid cultured 5J + 2C, R0040 + J23032,6O + 24B,
-
4. Cultured BBa_R0062 + BBa_I718008
+
*4.Extracted plasmid of 5J + 2C，J23032, B0034（strong RBS）,14G + BBa_B0015, R0040 + J23032, 6O + 24B, 6N + B0034 and then did digestion.
-
5. Digested pSB1C3, pSB1K3, pSB1A3, pSB1T3, BBa_R0062 + BBa_I718008, BBa_F2622 + BBa_CO261
+
*5.Digested cre, po，6O + 24B, B0034,2C.
-
6. Transformed Cre + BBa_B0015, pSB1A2, BBa_J04650
+
*6.Sent 6O + 24B, 5J + 2C to sequence, however all turned out to be wrong, so we need to religate them.
-
7. Transformed pSB1A2, BBa_J04650
+
*7.Sent 5J + 2C + PSB1C3，14G + BBa_B0015 + PSB1C3, R0040 + J23032, 6N + B0034 to sequence
-
8. Sent to sequence: BBa_R0062 + BBa_I718008, BBa_F2622 + BBa_B0015 → 1B0034
+
*8.Amplified  I746361 through PCR
-
9. Did gel extraction RBS + Cre + ssrA
+
-
9.8~9.14
+
===8.25~8.31===
-
1. Digested BBa_I13507, pSB1C3, BBa_R0040 + Po, BBa_R0040 + po + BBa_I13507
+
*1. Digested R0040 + J23032 and F2622 + BBa_B0015, B0034 + 2C, 6O + 24B
-
2. Incubated BBa_R0040→BBa_B0015
+
*2.Designed primer of APO
-
3. Assembled BBa_R0040 + po + BBa_I13507, BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + po + BBa_I13507
+
*3.Failed to PCR Cre + ssrA
-
4. Sent to sequence: BBa_B0034→BBa_B0015, BBa_R0040→BBa_B0015, BBa_R0040 + PO + BBa_I13507
+
*4.Transformed B0034 + 2C, R0040 + PO,
-
5.96-well plates experiment of BBa_R0040 + PO
+
*5.Recultured 6O + 24B, F2622 + BBa_CO261
-
6. Cultured BBa_R0040 + BBa_I13504 and observed its fluorescence
+
*6.Single digested F2622 + BBa_B0015,R0040 + po, compared with previous single digested BBa_B0015, but failed.
-
7. Transformed BBa_R0040 + BBa_J23032 + BBa_J04650, BBa_R0040 + Po + BBa_I13507
+
*7. Redigested  BBa_B0015, then did ligation and transformation.
 +
*8.Sent to sequence:  14G + BBa_B0015, R0040 + J23032, 6O + 24B, R0040 + po, 6N + B0034, B0034 + 2C
 +
*9.Recovered BBa_B0015 and  R0040 from conservation
-
9.15~9.21
+
==September==
-
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
+
===9.1~9.7===
-
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
+
*1. succeeded in amplifying Cre + ssrA by PCR
-
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
+
*2.Digested R0040, 6N + B0034, pSB1C3, pSB1K3, pSB1A3, pSB1T3, 6O + 24B, F2622 + BBa_CO261, Cre + BBa_B0015, 6N→BBa_B0015, B0034 + 14G + BBa_B0015, 4F + 6N, PCRed F2622 + BBa_B0015, F2622 + BBa_B0015 + R0040 + J23032,  6G, 24B
-
4. Prepared A +     and T +   plates
+
*3.ligated Cre + BBa_B0015, F2622 + BBa_CO261(pSB1C3), B0034 + 14G + BBa_B0015(pSB1T3), 6N + B0034 + 14G + BBa_B0015, *R0040 + J23032 + F2622 + BBa_B0015(pSB1T3), R0040 + po + I13507 (pSB1T3), RBS + Cre + ssrA + B0034 + 14G + BBa_B0015(pSB1T3) (C23 for short), RBS + Cre + ssrA (pSB1C3) (CreS for short), F2622 + BBa_B0015 (pSB1C3) (1223 for short), B0034 + 14G + BBa_B0015(pSB1C3), R0040 + BBa_I13504 (pSB1C3), I746361 + BBa_I13504 (pSB1C3),  R0040 → BBa_B0015 (pSB1C3), 6G + 24B (pSB1C3),  then transformed them.
-
5. Assembled BBa_I746361→BBa_B0015 + BBa_K145215 and BBa_R0040 + BBa_I13507, then did transformation
+
*4. Cultured 6O + 24B, B0034 + 2C
-
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
+
*5.Digested pSB1C3, pSB1K3, pSB1A3, pSB1T3, 6O + 24B, F2622 + BBa_CO261
-
7. Gel extraction of BBa_R0040 + Po + BBa_I13507
+
*6.Transformed Cre + BBa_B0015, 18F + 4F, 4F + 6N, 6N + B0034 + 14G + BBa_B0015, pSB1A2, K56G, Plac, C23, 2012K1220, K33L, K3J04650, J04650
-
8. Sent BBa_R0040 + BBa_J23032 + BBa_J04650 + BBa_K145215, BBa_I746361 + BBa_I13507 + BBa_R0040→BBa_B0015 to sequence
+
*7.Transformed pSB1A2, K56G, Plac, C23, 2012K1220, K33L, K3J04650, J04650
 +
*8.Sent to sequence: B0034 + 14G + BBa_B0015, 6N → BBa_B0015, B0034 + 2C, 6O + 24B,  12C + BBa_B0015 → 7F, F2622 + BBa_B0015 → 1B0034
 +
*9.Did gel extraction of 6N  + 19C + BBa_B0015, RBS + Cre + ssrA
-
9.22-9.28
+
===9.8~9.14===
-
1. Prepare LB incubate and LB plates
+
*1.Digested CreS, 1223, B0034 + 14G + BBa_B0015, 6N→BBa_B0015, I13507, pSB1C3, CreD, CreE, R0040 + po, R0040 + po + I13507, 6G + 3L
-
2. Incubated BBa_B0034→BBa_B0015, and then extracted plasmid
+
*2.Liquid cultured R0040→BBa_B0015, 6G + 24B
-
4. Digested BBa_I13521, BBa_I718008, BBa_B0034→BBa_B00155. Gel extraction of BBa_I718008, and BBa_I13507
+
*3. Ligated 6G + 3L, R0040 + po + I13507, R0040 + J23032 + J04650 ,R0040 + po + I13507, CreS, C23
-
6. Function test of BBa_I13521, BBa_I746361→BBa_B0015, BBa_I746361→ BBa_K145215, BBa_I746361 + BBa_I13507
+
*4.Sent to sequence: 1223, B0034→BBa_B0015, 6N→BBa_B0015, R0040→BBa_B0015, 6G + 3L, R0040 + po + I13507
-
7. PCR BBa_I13507
+
*5.Traditional assembly of Cre and C23
-
8. Assembled BBa_I718008 and BBa_B0034→BBa_B0015
+
*6.96-well plates experiment of R0040 + po
 +
*7.Cultured R0040 + BBa_I13504 and observed its fluorescence
 +
*8.Transformed R0040 + J23032 + J04650, 6G + 3L, R0040 + po + I13507
 +
===9.15~9.21===
 +
*1.Miniprep：I746361 + I13507, 6G + 3L, R0040 + J23032 + J04650, R0040 + Po + I13507,  I746361 + I13507, Cres + B0034 + 14G + BBa_B0015,  I746361 + I13507 + R0040→BBa_B0015, K145215 + R0040 + J23032 + J04650, I746361 + I13507 + R0040 + J23032 + F2622 + BBa_B0015 + K145215, R0040 + po + I13507, 6g,  I746361→BBa_B0015 + K145215, C23, I13521, K145215 + R0040 + J23032 + J04650
 +
*2.Digestion: I746361 + I13507, R0040 + J23032 + 12K, Cres, 5I + J23032 + J04650, Cre + B0034 + 14G + BBa_B0015,  I746361 + I13507 + R0040→BBa_B0015,K145215 + R0040 + J23032 + J04650,  R0040 + Po + I13507, R0040 + po + I13507, 6g,  I746361→BBa_B0015 + K145215
 +
*3.Liquid culture:  I746361 + I13507, Cres,  I746361 + I13507 + R0040→BBa_B0015, Cre + B0034 + 14G + BBa_B0015, cres + Po + I13507, 6G,  R0040 + J23032 + J04650, K145215 + R0040 + J23032 + J04650, R0040, K145215 + B0034 + J23032 + BBa_B0015, R0040 + Po + I13507
 +
*4. Prepared A +    and T +  plates
 +
*5. Ligated  I746361→BBa_B0015 + K145215 and R0040 + I13507,then did transformation
 +
*6. Function test: K145215 + R0040 + J23032 + J04650, R0040 + J23032 + J04650, R0040 + J23032 + J04650 + K145215, I13521,  I746361 + I13507,  I746361 + I13507 + R0040→BBa_B0015,  I746361 + 220 + R0040→BBa_B0015 + K145215, R0040 + Po + I13507
 +
*7. Gel extraction  of R0040 + po + I13507 and 6g
 +
*8. Sent 6G + 3L, R0040 + J23032 + J04650 + K145215,  I746361 + I13507 + R0040→BBa_B0015,  I746361 + 220 + R0040→BBa_B0015 + K145215 to sequence
 +
===9.22-9.28===
 +
*1.Resent  I746361 + 220 + R0040→BBa_B0015 + K145215 to sequence
 +
*2.Prepare LB liquid culture and LB plates
 +
*3.Liquid cultured 6G + R0040 + PO + I13507, C23,B0034→BBa_B0015 and 3L, 6G→I13507, and then extracted plasmid
 +
*4.Digested I13521、C23,B0034 + 14G + BBa_B0015、3L, cre、R0040 + po + I13507 + 6G、6G→I13507、B0034→BBa_B0015、B0034 + 14G + BBa_B0015 + psb1c3
 +
*5.Gel extraction of B0034 + 14G + BBa_B0015、3L、B0034 + 14G + BBa_B0015 + psb1c3、cre、3L、6G→I13507 and I13507
 +
*6.Function test of I13521、 I746361→BBa_B0015、 I746361→K145215、 I746361 + I13507
 +
*7.PCRI13507
 +
*8.Ligated cre and B0034→BBa_B0015
 +
*9.Transformed c23、B0034→BBa_B0015 and 3L
|}
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Latest revision as of 17:40, 1 October 2013

Animation


 

June

6.8~6.15

  • transformation of BBa_B0015，BBa_CO261 to psb1AK3 is successful
  • Transformed pSB1T3,pSB1K3, pSB1C3,pSB1A3, BBa_I13504 and BBa_I13504.
  • the bacteria precipitate transformed with pSB1C3 and pSB1A3 were red after centrifugation, the degree of red decreased in this order: pSB1K32 (pinkred) - pSB1A3 (pink) - pSB1A3(tinge) - pSB1K3(yellow).
  • Chloramphenicol was insoluble，and it was tested to be useless using DH5α.
  • prepare A + ,K + ,T + ,C + plates and antibiotic store liquor.
  • Digested and ligated BBa_CO261, pSB1C3,then transformed them
  • Digestion of BBa_I13504, BBa_B0015, and proved that the transformation was successful

6.16~6.22

  • 1.PCR to amplified 8B but failed
  • 2. Chloramphenicol was tested to be effective
  • 3.transformed pSB1T3, K2-8B BBa_CO261, BBa_B0015, BBa_I13504, pSB1K3(pSB1K3) and pSB1A3(pSB1A3)
  • 4.plasmid extraction of F2622 + BBa_CO261
  • 5.Digested F2622 + BBa_CO261 and pSB1T3 (EcoRⅠand PstⅠ)
  • 6.ligated F2622 + BBa_CO261, BBa_B0015 and pSB1T3

6.23~6.29

  • 1.strain conservation of F2622 + BBa_CO261, pSB1C3
  • 2.prepared ddH2O
  • 3.Added 1µl Dpn Ӏ into pSB1T3(pSB1T3) reaction system when digesting

July

6.30~7.6

  • 1.Digested F2622 + BBa_CO261 and pSB1T3(EcoRⅠand PstⅠ)
  • 2.ligated F2622 + BBa_CO261, BBa_I13504, and pSB1T3, but failed to transform

7.7~7.13

  • 1.plated F2622 + BBa_CO261 + BBa_I13504(1.2.3) and F2622 + BBa_CO261 + BBa_B0015(1.2.3) which were ligated 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 K2-3G(RBS + Cre), K5-24B, K5-R0040 and 3G, 24B were successful
  • 3.extracted plasmid of K2-8B, F2622 + BBa_CO261 and only K2-8B was successful
  • 4.Liquid cultured 24B, 3G, R0040, 6O, 7F, 12C and F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015, 4F.
  • 5. Successfully ligated F2622 + BBa_CO261 + BBa_I13504, 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, B0034M 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.ligated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015 with pSB1C3 carrier, then did transformation and liquid culture
  • 3.Liquid cultured R0040, 8B, 6O, F2622 + BBa_CO261, BBa_B0015
  • 4.Replated F2622 + BBa_CO261 + BBa_I13504, F2622 + BBa_CO261 + BBa_B0015
  • 5.Digested R0040, 6O, 24B
  • 6.Ligated 6O with 24B, then did transformation
  • 7.transformed 18O, 19C

August

8.3~8.10

  • 1.ligated 6N + 19C + BBa_B0015, after failing to ligate 6N(T7, promoter) with 19C(reporter)
  • 2.Liquid cultured 6O + 24B, and gel analysis of plasmid extraction and digestion products proved that the ligation was successful
  • 3.Transformed K3- I746361, K3-K145215, K2-15J and 5J + 2C. K145215 and 15J turned out to be a failure. Liquid cultured the rest.
  • 4.Digested 6O, 24B, pSB1C3, and ligated them overnight

8.11~8.17

  • 1.transformed 5J + 2C，18O, 19C， I746361,K145215,15J，K5-J23032，F2622 + BBa_CO261
  • 2.Extracted plasmid of K145215,15J,5J + 2C,F2622 + BBa_CO261
  • 3.Digested K145215，6N + I13507，BBa_CO261，5J + 2C,F2622 + BBa_CO261，
  • 4.Liquid cultured F2622，6N + 19C + BBa_B0015，J23032
  • 5.Sent K145215, 6N + I13507, F2622, 6N + 19C(PSN1C3), 6N + 19C + BBa_B0015(PSB1C3) to biotech company to sequence them.

8.18~8.24

  • 1.Ligated R0040 + J23032(Ptet + lock) ,B0034 + 2C , PO + LOX + R0040
  • 2.Transformed B0034 + 6N,6O + 24B,F2622 + BBa_CO261, R0040 + J23032
  • 3.Liquid cultured 5J + 2C, R0040 + J23032,6O + 24B,
  • 4.Extracted plasmid of 5J + 2C，J23032, B0034（strong RBS）,14G + BBa_B0015, R0040 + J23032, 6O + 24B, 6N + B0034 and then did digestion.
  • 5.Digested cre, po，6O + 24B, B0034,2C.
  • 6.Sent 6O + 24B, 5J + 2C to sequence, however all turned out to be wrong, so we need to religate them.
  • 7.Sent 5J + 2C + PSB1C3，14G + BBa_B0015 + PSB1C3, R0040 + J23032, 6N + B0034 to sequence
  • 8.Amplified I746361 through PCR

8.25~8.31

  • 1. Digested R0040 + J23032 and F2622 + BBa_B0015, B0034 + 2C, 6O + 24B
  • 2.Designed primer of APO
  • 3.Failed to PCR Cre + ssrA
  • 4.Transformed B0034 + 2C, R0040 + PO,
  • 5.Recultured 6O + 24B, F2622 + BBa_CO261
  • 6.Single digested 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: 14G + BBa_B0015, R0040 + J23032, 6O + 24B, R0040 + po, 6N + B0034, B0034 + 2C
  • 9.Recovered BBa_B0015 and R0040 from conservation

September

9.1~9.7

  • 1. succeeded in amplifying Cre + ssrA by PCR
  • 2.Digested R0040, 6N + B0034, pSB1C3, pSB1K3, pSB1A3, pSB1T3, 6O + 24B, F2622 + BBa_CO261, Cre + BBa_B0015, 6N→BBa_B0015, B0034 + 14G + BBa_B0015, 4F + 6N, PCRed F2622 + BBa_B0015, F2622 + BBa_B0015 + R0040 + J23032, 6G, 24B
  • 3.ligated Cre + BBa_B0015, F2622 + BBa_CO261(pSB1C3), B0034 + 14G + BBa_B0015(pSB1T3), 6N + B0034 + 14G + BBa_B0015, *R0040 + J23032 + F2622 + BBa_B0015(pSB1T3), R0040 + po + I13507 (pSB1T3), RBS + Cre + ssrA + B0034 + 14G + BBa_B0015(pSB1T3) (C23 for short), RBS + Cre + ssrA (pSB1C3) (CreS for short), F2622 + BBa_B0015 (pSB1C3) (1223 for short), B0034 + 14G + BBa_B0015(pSB1C3), R0040 + BBa_I13504 (pSB1C3), I746361 + BBa_I13504 (pSB1C3), R0040 → BBa_B0015 (pSB1C3), 6G + 24B (pSB1C3), then transformed them.
  • 4. Cultured 6O + 24B, B0034 + 2C
  • 5.Digested pSB1C3, pSB1K3, pSB1A3, pSB1T3, 6O + 24B, F2622 + BBa_CO261
  • 6.Transformed Cre + BBa_B0015, 18F + 4F, 4F + 6N, 6N + B0034 + 14G + BBa_B0015, pSB1A2, K56G, Plac, C23, 2012K1220, K33L, K3J04650, J04650
  • 7.Transformed pSB1A2, K56G, Plac, C23, 2012K1220, K33L, K3J04650, J04650
  • 8.Sent to sequence: B0034 + 14G + BBa_B0015, 6N → BBa_B0015, B0034 + 2C, 6O + 24B, 12C + BBa_B0015 → 7F, F2622 + BBa_B0015 → 1B0034
  • 9.Did gel extraction of 6N + 19C + BBa_B0015, RBS + Cre + ssrA

9.8~9.14

  • 1.Digested CreS, 1223, B0034 + 14G + BBa_B0015, 6N→BBa_B0015, I13507, pSB1C3, CreD, CreE, R0040 + po, R0040 + po + I13507, 6G + 3L
  • 2.Liquid cultured R0040→BBa_B0015, 6G + 24B
  • 3. Ligated 6G + 3L, R0040 + po + I13507, R0040 + J23032 + J04650 ,R0040 + po + I13507, CreS, C23
  • 4.Sent to sequence: 1223, B0034→BBa_B0015, 6N→BBa_B0015, R0040→BBa_B0015, 6G + 3L, R0040 + po + I13507
  • 5.Traditional assembly of Cre and C23
  • 6.96-well plates experiment of R0040 + po
  • 7.Cultured R0040 + BBa_I13504 and observed its fluorescence
  • 8.Transformed R0040 + J23032 + J04650, 6G + 3L, R0040 + po + I13507

9.15~9.21

  • 1.Miniprep：I746361 + I13507, 6G + 3L, R0040 + J23032 + J04650, R0040 + Po + I13507, I746361 + I13507, Cres + B0034 + 14G + BBa_B0015, I746361 + I13507 + R0040→BBa_B0015, K145215 + R0040 + J23032 + J04650, I746361 + I13507 + R0040 + J23032 + F2622 + BBa_B0015 + K145215, R0040 + po + I13507, 6g, I746361→BBa_B0015 + K145215, C23, I13521, K145215 + R0040 + J23032 + J04650
  • 2.Digestion: I746361 + I13507, R0040 + J23032 + 12K, Cres, 5I + J23032 + J04650, Cre + B0034 + 14G + BBa_B0015, I746361 + I13507 + R0040→BBa_B0015,K145215 + R0040 + J23032 + J04650, R0040 + Po + I13507, R0040 + po + I13507, 6g, I746361→BBa_B0015 + K145215
  • 3.Liquid culture: I746361 + I13507, Cres, I746361 + I13507 + R0040→BBa_B0015, Cre + B0034 + 14G + BBa_B0015, cres + Po + I13507, 6G, R0040 + J23032 + J04650, K145215 + R0040 + J23032 + J04650, R0040, K145215 + B0034 + J23032 + BBa_B0015, R0040 + Po + I13507
  • 4. Prepared A + and T + plates
  • 5. Ligated I746361→BBa_B0015 + K145215 and R0040 + I13507,then did transformation
  • 6. Function test: K145215 + R0040 + J23032 + J04650, R0040 + J23032 + J04650, R0040 + J23032 + J04650 + K145215, I13521, I746361 + I13507, I746361 + I13507 + R0040→BBa_B0015, I746361 + 220 + R0040→BBa_B0015 + K145215, R0040 + Po + I13507
  • 7. Gel extraction of R0040 + po + I13507 and 6g
  • 8. Sent 6G + 3L, R0040 + J23032 + J04650 + K145215, I746361 + I13507 + R0040→BBa_B0015, I746361 + 220 + R0040→BBa_B0015 + K145215 to sequence


9.22-9.28

  • 1.Resent I746361 + 220 + R0040→BBa_B0015 + K145215 to sequence
  • 2.Prepare LB liquid culture and LB plates
  • 3.Liquid cultured 6G + R0040 + PO + I13507, C23,B0034→BBa_B0015 and 3L, 6G→I13507, and then extracted plasmid
  • 4.Digested I13521、C23,B0034 + 14G + BBa_B0015、3L, cre、R0040 + po + I13507 + 6G、6G→I13507、B0034→BBa_B0015、B0034 + 14G + BBa_B0015 + psb1c3
  • 5.Gel extraction of B0034 + 14G + BBa_B0015、3L、B0034 + 14G + BBa_B0015 + psb1c3、cre、3L、6G→I13507 and I13507
  • 6.Function test of I13521、 I746361→BBa_B0015、 I746361→K145215、 I746361 + I13507
  • 7.PCRI13507
  • 8.Ligated cre and B0034→BBa_B0015
  • 9.Transformed c23、B0034→BBa_B0015 and 3L


 
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