NYMU Taipei/Benchman

From 2007.igem.org

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 Oct 03, 07
pOmpC (P.E. #3) plasmid extraction #3 2,271     0.21 1.42 1.12 Oct 03, 07
pOmpC (P.E. #4) plasmid extraction #4 2,271     0.25 1.37 1.03 Oct 03, 07
pOmpC (D. SP) digestion (S. P.) as back vector, #4 selected 2,253   0.14 0.01 n/a n/a Oct 04, 07
TATA_INSA (D. XP) digestion (X. P.) as back insert  193   0.25 0.02 n/a 0.29 Oct 03, 07
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 Oct 10, 07
TATB_INSB (D. XP) digestion (X. P.) as back insert 220     n/a n/a n/a 10, 07
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 Oct 03, 07
CinR+HSL+D-term (P.E.) plasmid extraction #1, #3, #4 (wrong!!!)  4,804     0.41, 0.25, 0.41     ">Sep 27, 07
CinR+HSL+D-term (D. E) first digestion (E.) as front vector (wrong!!!) 4,804   0.19       Oct 03, 07
CinR+HSL+D-term (D. EX) second digestion (X.) as front vector (wrong!!!) 4,804   0.19 0.02 n/a 0.13 Oct 04, 07
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 Oct 03, 07
pCinRHSL (P.E. #2) plasmid extraction #2       0.29 1.58 1.26 Oct 03, 07
pCinRHSL (D. SP) digestion (S. P.) as back vector, #1 selected     0.17 0.03 n/a 0.18 Oct 04, 07
OmpRBS (D. XP) digestion (X. P.) as back insert     0.24 0.01 n/a n/a Oct 03, 07
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.

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