NYMU Taipei/Benchman
From 2007.igem.org
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<td align="right"><font size="1">1.30</font></td> | <td align="right"><font size="1">1.30</font></td> | ||
<td align="right"><font size="1">0.97</font></td> | <td align="right"><font size="1">0.97</font></td> | ||
| - | <td align="right"><font size="1"> | + | <td align="right"><font size="1">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3 Oct 03, 07]</font></td> |
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
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<h3>Notes</h3> | <h3>Notes</h3> | ||
<ul> | <ul> | ||
Revision as of 14:46, 24 October 2007
OD and GEL checks
| Part name |
Type and Description |
length (bp) | PCR check | weight (g) | concentration (ug/uL) | A260/A280 | A260/A230 | date |
| pOmpC (P.E. #1) | plasmid extraction #1 | 2,271 | 0.20 | 1.30 | 0.97 | Oct 03, 07 | ||
| pOmpC (P.E. #2) | plasmid extraction #2 | 2,271 | 0.19 | 1.33 | 1.00 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | ||
| pOmpC (P.E. #3) | plasmid extraction #3 | 2,271 | 0.21 | 1.42 | 1.12 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | ||
| pOmpC (P.E. #4) | plasmid extraction #4 | 2,271 | 0.25 | 1.37 | 1.03 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | ||
| pOmpC (D. SP) | digestion (S. P.) as back vector, #4 selected | 2,253 | 0.14 | 0.01 | n/a | n/a | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_4">Oct 04, 07</a> | |
| TATA_INSA (D. XP) | digestion (X. P.) as back insert | 193 | 0.25 | 0.02 | n/a | 0.29 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | |
| TATA_INSA (PCR) | PCR for TATA + INSA | 209 | 0.24; 0.30 | Oct 9, 07 | ||||
| TATB_INSB (PCR) | PCR for TATB + INSB | 233 | 0.35; 0.35 | Oct 9, 07 | ||||
| TATA_INSA (D. XP) | digestion (X. P.) as back insert | 193 | n/a | n/a | n/a | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_10">Oct 10, 07</a> | ||
| TATB_INSB (D. XP) | digestion (X. P.) as back insert | 220 | n/a | n/a | n/a | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_10">Oct 10, 07</a> | ||
| pOmpC (D. SP) | digestion (S. P.) as back vector, #1 selected | 2,253 | n/a | n/a | n/a | Oct 11, 07 | ||
| pOmpC + TATA_INSA | back ligation pOmpC (D. SP) + TATA_INSA (D. XP) | 2,454 | 430, 613 | Oct 13, 07 & Oct 16,07 | ||||
| pOmpC+TATA_INSA (D. SP) | digestion (S. P.) as back vector | 2,436 2,427 | ||||||
| pOmpC+TATA_INSA + TATB_INSB | back ligation pOmpC+TATA_INSA (D. SP) + TATB_INSB (D. XP) | 2,655 | 613, 814 | |||||
| TATB_INSB (D. ES) | digestion (E. S.) as front insert (wrong!!!) | 0.27 | 0.03 | n/a | 0.21 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | ||
| CinR+HSL+D-term (P.E.) | plasmid extraction #1, #3, #4 (wrong!!!) | 4,804 | 0.41, 0.25, 0.41 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_27#CinR.2BHSL.2BD-term_check">Sep 27, 07</a> | ||||
| CinR+HSL+D-term (D. E) | first digestion (E.) as front vector (wrong!!!) | 4,804 | 0.19 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | ||||
| CinR+HSL+D-term (D. EX) | second digestion (X.) as front vector (wrong!!!) | 4,804 | 0.19 | 0.02 | n/a | 0.13 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_4">Oct 04, 07</a> | |
| TATB_INSB + CinR+HSL+D-term | front ligation TATB_INSB (D. ES) + CinR+HSL+D-term (D. EX) (wrong!!!) | Oct 05, 07 | ||||||
| pOmpC+TATA_INSA+TATB_INSB (D. SP) | digestion (S.P.) as back vector | 2,637 | ||||||
| CinR+HSL+D-term (D. XP) | digestion (X.P.) as back insert | 1,647 | ||||||
| system 1 | ligation pOmpC+TATA_INSA+TATB_INSB + CinR+HSL+D-term | 4,292 | 814, 2,451 | |||||
| pCinRHSL (P.E. #1) | plasmid extraction #1 | 0.23 | 1.70 | 1.49 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | |||
| pCinRHSL (P.E. #2) | plasmid extraction #2 | 0.29 | 1.58 | 1.26 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | |||
| pCinRHSL (D. SP) | digestion (S. P.) as back vector, #1 selected | 0.17 | 0.03 | n/a | 0.18 | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_4">Oct 04, 07</a> | ||
| OmpRBS (D. XP) | digestion (X. P.) as back insert | 0.24 | 0.01 | n/a | n/a | <a href="https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_3">Oct 03, 07</a> | ||
| pCinRHSL+OmpRBS | ligation pCinRHSL (D. SP) + OmpRBS (D. XP) | Oct 05, 07 | ||||||
| pCinRHSL+OmpRBS (D. SP) | digestion (S. P.) pCinRHSL+OmpRBS as back vector (a bit smear) | 2,443 | 0.14 (too few) | Oct 9, 07 | ||||
| pCinRHSL+OmpRBS (plasmid) | plasmid extraction of pCinRHSL+OmpRBS (clone 7 and clone 10 on the plate) after liquid culture | 0.020; 0.019 | 1.95; 1.83 | 2.18; 2.31 | Oct 10, 07 | |||
| pCinRHSL+OmpRBS (D. SP) | digestion (S. P.) pCinRHSL+OmpRBS as back vector (smear) | 2,443 | Oct 10, 07 | |||||
| pCinRHSL+OmpRBS (D. SP) | digestion (S. P.) pCinRHSL+OmpRBS as back vector using plasmid from clone 7 | 2,443 | 0.05 | n/a | 0.22 | Oct 11, 07 | ||
| TATD_IDE (D. XP) | digestion (X. P.) as back insert | 3,190 | 0.10 (too few) | Oct 9, 07 | ||||
| TATD + IDE (PCR) | re-PCR for TATD + IDE | 3,203 | Oct 10, 07 | |||||
| TATD_IDE (D. XP) | digestion (X. P.) as back insert | 3,190 | Oct 11, 07 | |||||
| pCinRHSL+OmpRBS + TATD_IDE | ligation pCinRHSL+OmpRBS + TATD_IDE | |||||||
| pCinRHSL+OmpRBS+TATD_IDE (D. SP) | digestion (S. P.) as back vector | |||||||
| D-term (D. XP) | digestion (X. P.) as back insert | |||||||
| system 2 | ligation pCinRHSL+OmpRBS+TATD_IDE + D-term |
Notes
- A260/A280 should be around 1.70
- A260/A230 should be less than 3.00
- color key
- problemistic (read)
- finished (yellow)
- await (blue)
- digestion and buffer
- SepI and PstI (SP) digestion using buffer 2, creates back vector
- XbaI and PstI (XP) digestion using buffer 3, creates back insert
- Assembly process
- STEP 1. prepare insert and vector by digestion
- STEP 2. check digestion by GEL separation
- if band is clear at correct position (length), cut the gel and purify/extract it
- else go back to STEP 1.
- STEP 3. ligate insert with vector, transform it into competent cell, and extract plasmid from liquid culture
- STEP 4. check ligation by VF2, VR PCR with GEL separation
- if band is clear at correct position (length), cut the gel and purify/extract it (ligation is done)
- else go back to STEP 3.
