"
SCU Weekly
From 2013.igem.org
(Difference between revisions)
Zhangbingbio (Talk | contribs) |
Zhangbingbio (Talk | contribs) |
||
| (5 intermediate revisions not shown) | |||
| Line 25: | Line 25: | ||
| | ||
{| style="text-align:justify; text-justify:inter-ideograph; width: 800px" border="0" cellspacing="1" cellpadding="20" align="center" | {| style="text-align:justify; text-justify:inter-ideograph; width: 800px" border="0" cellspacing="1" cellpadding="20" align="center" | ||
| + | |- | ||
| | | | ||
| - | |||
| - | |||
| - | |||
---- | ---- | ||
| - | + | ==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 | ||
|} | |} | ||
__NOEDITSECTION__ | __NOEDITSECTION__ | ||
Latest revision as of 17:40, 1 October 2013
June6.8~6.15
6.16~6.22
6.23~6.29
July6.30~7.6
7.7~7.13
7.14~7.201.no much experiment 7.21~7.27
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
August8.3~8.10
8.11~8.17
8.18~8.24
8.25~8.31
September9.1~9.7
9.8~9.14
9.15~9.21
9.22-9.28
|
