Day 25. 2008/04/14
Today’s all results were strange. The plasmids extracted by Kit not so pure, because there were two bands stay at near positions where my plasmid should be. Although I took about four hours to digest, of the five samples I only got one MAY BE what I want.
So that tomorrow I will have to extract again, by hand and Kit respectively.
Today’s all results were strange. The plasmids extracted by Kit not so pure, because there were two bands stay at near positions where my plasmid should be. Although I took about four hours to digest, of the five samples I only got one MAY BE what I want.
So that tomorrow I will have to extract again, by hand and Kit respectively.
Day 26. 2008/04/15
I extracted the plasmids by Kit, but the photo was still not very perfect (080415-PLASMID.jpg). Maybe my gel had something incorrect.
XhoI digestion was not good too. When I checked my primers in the evening, I got very surprised. My sense primer not concludes XhoI site, but EcoRI and NcoI!
Now, the problem got solved.
I extracted the plasmids by Kit, but the photo was still not very perfect (080415-PLASMID.jpg). Maybe my gel had something incorrect.
XhoI digestion was not good too. When I checked my primers in the evening, I got very surprised. My sense primer not concludes XhoI site, but EcoRI and NcoI!
Now, the problem got solved.
Day 27. 2008/04/16
Digestion 1 (I got the enzymes a wrong quality, which should be 0.5, but I added 1, so the concentration was wrong):
B1 & R1: XhoI 1; BamHI 1; 10×K Buffer 1; ddH2O 3; plasmid 5;
B2 & R2: XhoI 1; NcoI 1; 10×K Buffer 1; 10×BSA 1; ddH2O 2; plasmid 5;
R3 (only a little test for another two sites on pMD-18): EcoRI 0.5; HindIII 0.5; 10×M Buffer 1; ddH2O 3; plasmid 5;
But the cut bands were so faint that I couldn’t see it well without Photoshop editing (080416-digestion1.jpg). I guessed it was because of that I added more plasmids.
So…
Digestion 2
B1 & R1: XhoI 0.5; BamHI 0.5; 10×K Buffer 1; ddH2O 6; plasmid 2;
B2 & R2: XhoI 0.5; NcoI 0.5; 10×K Buffer 1; 10×BSA 1; ddH2O 5; plasmid 2;
R3 (only a little test for another two sites on pMD-18): EcoRI 0.5; HindIII 0.5; 10×M Buffer 1; dd H2O 5; plasmid 2;
This time the bands were little lighter (I only detected two of them), but still faint. So I left the other tubes to digest overnight.
Waiting for tomorrow’s electrophoresis.
Digestion 1 (I got the enzymes a wrong quality, which should be 0.5, but I added 1, so the concentration was wrong):
B1 & R1: XhoI 1; BamHI 1; 10×K Buffer 1; ddH2O 3; plasmid 5;
B2 & R2: XhoI 1; NcoI 1; 10×K Buffer 1; 10×BSA 1; ddH2O 2; plasmid 5;
R3 (only a little test for another two sites on pMD-18): EcoRI 0.5; HindIII 0.5; 10×M Buffer 1; ddH2O 3; plasmid 5;
But the cut bands were so faint that I couldn’t see it well without Photoshop editing (080416-digestion1.jpg). I guessed it was because of that I added more plasmids.
So…
Digestion 2
B1 & R1: XhoI 0.5; BamHI 0.5; 10×K Buffer 1; ddH2O 6; plasmid 2;
B2 & R2: XhoI 0.5; NcoI 0.5; 10×K Buffer 1; 10×BSA 1; ddH2O 5; plasmid 2;
R3 (only a little test for another two sites on pMD-18): EcoRI 0.5; HindIII 0.5; 10×M Buffer 1; dd H2O 5; plasmid 2;
This time the bands were little lighter (I only detected two of them), but still faint. So I left the other tubes to digest overnight.
Waiting for tomorrow’s electrophoresis.
Day 28. 2008/04/17
The overnight digestion wasn’t more ideal than 1-2 hours reaction. I even couldn’t find any bands on the gel!
Sure, that’s only a test, what I should to do was to extract enough plasmids, and this time I also used the Kit.
But…
Oh! My goodness! I forgot to change the spin column onto another EP tube, so I had to precipitate plasmid DNA by ethanol that afternoon. Good luck, the result was not bad.
Then, from my tutor I got 10μl of E. coli which including the expression plasmid pET30a, and also 1.5μl of pure plasmid pET30a and pGEX6-1. I transferred the 10μl bacteria into liquid LB with Kan.
The overnight digestion wasn’t more ideal than 1-2 hours reaction. I even couldn’t find any bands on the gel!
Sure, that’s only a test, what I should to do was to extract enough plasmids, and this time I also used the Kit.
But…
Oh! My goodness! I forgot to change the spin column onto another EP tube, so I had to precipitate plasmid DNA by ethanol that afternoon. Good luck, the result was not bad.
Then, from my tutor I got 10μl of E. coli which including the expression plasmid pET30a, and also 1.5μl of pure plasmid pET30a and pGEX6-1. I transferred the 10μl bacteria into liquid LB with Kan.
Day 29. 2008/04/18
I did a 100μl digestion for later gel extraction, (080418-digestion2-100ul.jpg).
The pET30a bacteria didn’t grow! I added some LB into the original EP tube, swung for 1 hour, then transferred into antibiotic free LB.
I did a 100μl digestion for later gel extraction, (080418-digestion2-100ul.jpg).
The pET30a bacteria didn’t grow! I added some LB into the original EP tube, swung for 1 hour, then transferred into antibiotic free LB.
Day 30. 2008/04/19
The antibiotic free LB got some bacteria, but LB-Kan didn’t turn cloudy, so I had to transfer the pure plasmids into E. coli.
Maybe later I will try to make competent cells, so I needed to preserve some plasmid free cells. I added 35μl competent cell into the pGEX6-1 and pET30a vectors respectively, and the last 30μl of cell I transferred into antibiotic free liquid LB.
The antibiotic free LB got some bacteria, but LB-Kan didn’t turn cloudy, so I had to transfer the pure plasmids into E. coli.
Maybe later I will try to make competent cells, so I needed to preserve some plasmid free cells. I added 35μl competent cell into the pGEX6-1 and pET30a vectors respectively, and the last 30μl of cell I transferred into antibiotic free liquid LB.