IGEM:NYMU/2007/Benchman

h3>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

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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.