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JRH4712-63 (1)JRH4712-64 (2)
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Lab Book Ref: JRH4712-64
Ligation reactions were set up as follows:
The reactions were placed in the cold room at 4 °C overnight.
Digestion product and pBad/His are at reasonably even concentrations.
Ligation reactions were set up as follows:
| - | Reaction x 3 | +ve control |
| Digestion product (4712/63) | 4 μL | 0 μL |
| pBAD/His | 4 μL | 4 μL |
| Water | 0 μL | 4 μL |
| Ligase buffer | 1 μL | 1 μL |
| Ligase | 1 μL | 1 μL |
The reactions were placed in the cold room at 4 °C overnight.
Digestion product and pBad/His are at reasonably even concentrations.
I am perhaps the worlds biggest wally!
I will not be doing the experiment listed above as I know that if it works it will be wrong anyway.
I used the wrong enzymes when I did the digestion as I put in restriction sites for NcoI and XhoI not EcoR1 as that cuts inside the gene. My mistake was not apparent on the gel of the digestion as the two fragments obtained are 3019 and 56. 3019 and 3075 appear at the same place on a 1% ordinary agarose gel. in the same way as the previous problem with the β-glucuronidase PCR product and digestion product appearing to run the same amount.
I also cannot ligate (once I have correctly digested) into my main stock of pBAD/His as that is also cut with EcoR1 and Nco1.
Tomorrow I will take a stock of ordinary pBAD/His and the β-galactosidase PCR product and do a digestion of them with the correct enzymes.
Also the +ve control would be nonsense and not work as it will not ligate without the presence of an insert as it has been cut at two sites not one.
However it is good that I have spotted this problem now and not later when I would be confused as to why the protein is wrong/transformation hasn't worked, though frustrating that I made such a fundamental error in the first place.
;I will not be doing the experiment listed above as I know that if it works it will be wrong anyway.
I used the wrong enzymes when I did the digestion as I put in restriction sites for NcoI and XhoI not EcoR1 as that cuts inside the gene. My mistake was not apparent on the gel of the digestion as the two fragments obtained are 3019 and 56. 3019 and 3075 appear at the same place on a 1% ordinary agarose gel. in the same way as the previous problem with the β-glucuronidase PCR product and digestion product appearing to run the same amount.
I also cannot ligate (once I have correctly digested) into my main stock of pBAD/His as that is also cut with EcoR1 and Nco1.
Tomorrow I will take a stock of ordinary pBAD/His and the β-galactosidase PCR product and do a digestion of them with the correct enzymes.
Also the +ve control would be nonsense and not work as it will not ligate without the presence of an insert as it has been cut at two sites not one.
However it is good that I have spotted this problem now and not later when I would be confused as to why the protein is wrong/transformation hasn't worked, though frustrating that I made such a fundamental error in the first place.