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Team:University of Ottawa/10 July 2008
From 2008.igem.org
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:'''Digestion of PTP2 Ligation Product''' | :'''Digestion of PTP2 Ligation Product''' | ||
::<li> Another digestion of PTP2 was attempted using EcoRI - sadly I did not get the bands I needed, it seems the PTP2 was not correctly integrated into the Vector. | ::<li> Another digestion of PTP2 was attempted using EcoRI - sadly I did not get the bands I needed, it seems the PTP2 was not correctly integrated into the Vector. | ||
| + | '''Chris''' | ||
| + | :'''Ligation of AtCRE''' | ||
| + | ::<li> ligated AtCRE with 1 uL T4 ligase, 5 uL buffer and 1 uL enzyme. | ||
| + | ::<li> encountered issues with protocol and communication; standard ligation not exactly followed | ||
| + | ::<li> denatured ligase at 65 C for 10 minutes | ||
| + | :'''PCR Amplification of AtCRE''' | ||
| + | ::<li> followed Phusion high fidelity PCR protocol | ||
| + | ::<li> forgot to dilute primers tenfold | ||
| + | ::<li> prepared a 1% gel for confirmation of AtCRE; ran out of time to run gel. | ||
Revision as of 19:45, 16 July 2008
Contents |
Today in the lab
Dan
- Gel of S&D&T PCR products
- The bands I needed were on the gel, they were somewhat close to non specific binding bands, so I took extra caution when cutting them out.
- After using the gel extraction kit the obtained concentrations were decent.
- PCR amplification of S&D&T
- Performed PCR on the S&D&T obtained products, if it works, It would be easy to obtain more DNA and would save the hassle of going back to step 1.
- Atcre and construct 1 concentrations
- Both these DNA fragments resulted in very low concentrations following gel extraction. Chris went ahead and used Atcre anyways. The concentration of construct 1 was too low to use.
- Talked to Cory and realized that AgeI could be used instead of ClaI. That would solve our problem of performing ligation on a 2bp overhang.
- We ordered new primers today for this.
Looking back
- After Matt encountered problems with reduced enzymatic activity of restriction enzymes, I decided to redigest and reconfirm
Matt
- PCR confirmation
- Tested absorbance and Ran a PCR confirmation of IP producing cells (97 plasmid int. in BY4742 yeast strain) using both wild type BY4742 and H20 as control's.
- Digestion of PTP2 Ligation Product
- Another digestion of PTP2 was attempted using EcoRI - sadly I did not get the bands I needed, it seems the PTP2 was not correctly integrated into the Vector.
Chris
- Ligation of AtCRE
- ligated AtCRE with 1 uL T4 ligase, 5 uL buffer and 1 uL enzyme.
- encountered issues with protocol and communication; standard ligation not exactly followed
- denatured ligase at 65 C for 10 minutes
- PCR Amplification of AtCRE
- followed Phusion high fidelity PCR protocol
- forgot to dilute primers tenfold
- prepared a 1% gel for confirmation of AtCRE; ran out of time to run gel.
