NYMU Taipei/Lab Notes/2007 9 11
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*** insert (3 uL) vs. vector (1 uL) at 09/10, 2007: the quantity are 18ug = 18,000 ng vs. 13ug = 13,000 ng (original measure) | *** insert (3 uL) vs. vector (1 uL) at 09/10, 2007: the quantity are 18ug = 18,000 ng vs. 13ug = 13,000 ng (original measure) | ||
*** a trivial failure (absent of vector part) at 09/08, 2007 | *** a trivial failure (absent of vector part) at 09/08, 2007 | ||
| - | ** According to YEA ligation protocol, it requires | + | ** According to [http://www.yeastern.com/index.html YEA (益生)] ligation protocol, it requires |
*** ligation buffer A 1uL | *** ligation buffer A 1uL | ||
| - | *** ligation buffer B 1uL | + | *** ligation buffer B 1uL (Note: only needed in blunt end ligation) |
*** vector 50 ng or 100 ng (proposed by pett) | *** vector 50 ng or 100 ng (proposed by pett) | ||
*** insert 300 ng | *** insert 300 ng | ||
*** ligase 1uL | *** ligase 1uL | ||
*** add water to make total volume as 10 uL | *** add water to make total volume as 10 uL | ||
| + | ** Because the mistake on calculation of part concentrations, the concentration is over-estimated (concentration is far lower than we expected) | ||
| + | *** Obviously, the concentration of INS_A from 惇茹 (9/8, 2007) is too low | ||
| + | *** Thus, we use GEL to check the 海威's (9/5, 2007) PCR product (10uL), the band seems good | ||
| + | * Remark | ||
| + | ** the key of failure might be the insert concentration is too low | ||
| + | ** the corrected concentration of pOmpC (vector) should be 0.06 ug/uL (60ng/uL) and INS_A (insert) should be 0.13 ug/uL (130ng/uL) | ||
| + | ** to fulfill the requirement of ligation protocol, the volume of vector should be 1.66 uL and volume of insert should be 2.3 uL | ||
| + | ** However, we do not believe the concentration is correct and it might be very low (because the OD is almost zero) | ||
| + | ** To correctly measure the concentration and increase the production of INS_A through PCR, GEL extraction and digestion are two ways to successful construct | ||
| + | ** PCR might be tested and varied by different annealing temperature | ||
| + | ** Beside, our vector (pOmpC) has been successfully ligated with other Biobrick. It should be O.K. (However, we use ligation kit from different company) | ||
Latest revision as of 08:39, 14 September 2007
- Fresh back
- two different PCR (INS_A and INS_B) have been done by 海威 (9/5, 2007) and 惇茹 (9/8, 2007)
- we had failed three times for construction of pOmpC + INS_A using 惇茹's INS_A and following three different ligation protocol
- insert is INS_A and vector is pOmpC
- insert (1 uL) vs. vector (1 uL) at 09/09, 2007: the quantity are 6ug = 6,000 ng vs. 13ug = 13,000 ng (original measure)
- insert (2 uL) vs. vector (1 uL) at 09/10, 2007: the quantity are 12ug = 12,000 ng vs. 13ug = 13,000 ng (original measure)
- insert (3 uL) vs. vector (1 uL) at 09/10, 2007: the quantity are 18ug = 18,000 ng vs. 13ug = 13,000 ng (original measure)
- a trivial failure (absent of vector part) at 09/08, 2007
- According to [http://www.yeastern.com/index.html YEA (益生)] ligation protocol, it requires
- ligation buffer A 1uL
- ligation buffer B 1uL (Note: only needed in blunt end ligation)
- vector 50 ng or 100 ng (proposed by pett)
- insert 300 ng
- ligase 1uL
- add water to make total volume as 10 uL
- Because the mistake on calculation of part concentrations, the concentration is over-estimated (concentration is far lower than we expected)
- Obviously, the concentration of INS_A from 惇茹 (9/8, 2007) is too low
- Thus, we use GEL to check the 海威's (9/5, 2007) PCR product (10uL), the band seems good
- Remark
- the key of failure might be the insert concentration is too low
- the corrected concentration of pOmpC (vector) should be 0.06 ug/uL (60ng/uL) and INS_A (insert) should be 0.13 ug/uL (130ng/uL)
- to fulfill the requirement of ligation protocol, the volume of vector should be 1.66 uL and volume of insert should be 2.3 uL
- However, we do not believe the concentration is correct and it might be very low (because the OD is almost zero)
- To correctly measure the concentration and increase the production of INS_A through PCR, GEL extraction and digestion are two ways to successful construct
- PCR might be tested and varied by different annealing temperature
- Beside, our vector (pOmpC) has been successfully ligated with other Biobrick. It should be O.K. (However, we use ligation kit from different company)
