Day 15. 2008/04/04
What?! This noon I found some colonies on my plates, of which both the new X-gal spread and the more old X-gal spread, showed blue color! So that it must be that the former X-gal had lost its activity, and finally, I saw the beautiful and lovely blue of the colonies!
The Amp-free plates got a lot of colonies, contrary, there were almost not colonies on the Amp plates. There were nothing on the empty transferred plate; the pUC-18 plate got the full plate of tiny colonies. They all verified that my transformation was succeed; however, there are a few colonies on the radish and rape ligation plate, showed that most of the bacteria died on the Amp plates. Was there anything wrong in my last ligation? If it were, I would say that I got a deficient ligation time, which at least one hour but I only maintained 45 minuets at most… but even though it may be wrong in the ligation time, the un-ligated empty T-vectors should be transferred into cells, then show lots of colonies on the plates, just like the last time… well, the more I thought, the more I got puzzled.
At last, I make four new plate, with two Amp-free and two Amp added, each of them was covered by X-gal and IPTG (40:16). I spread all my remained bacteria liquid, including the liquid of this and the last time, onto the two Amp-free plates after 1.5 hours’ swing. On each Amp-added plates, I transferred 8×5 colonies from my last plates, one radish plate and one rape plate. I wanted to find some recons from the last plates. Then I transferred four single colonies from the new grown colonies, one blue from radish, two white from radish, and one white from rape, into four test tubes, then I swung them overnight for tomorrow’s PCR.
Ok, hoping to see recons, I also want to try endonuclease digesting earlier!
Day 16. 2008/04/05
This morning, I got out my plates. Now I got some new colonies which came from my second transformation. They are almost white, but it can’t instead of a succeed transformation, for some may be fake positive.
I did a colony PCR, used the bacteria liquid I swung yesterday.
I collected some colonies from the two Amp-free plates, transferred the colonies into 8 test tubes, radish tubes and 4 rape tubes. Then the PCR result showed that two of the four samples contained my target fragment, (electrophoresis photo: 080405-ColonyPCR.jpg). So that I see a beam of light toward the next step. Once again, I transferred another 12 (or 13?) single colonies into test tubes.
Ok, tomorrow, another batch of colony PCR!
Here I have a strange problem, which about plasmid extraction reagent. Solution I is a mixture of glucose, EDTA, and Tris-HCl, all the manual say that it should be autoclave sterilized. Glucose is unstable in high temperature, so its sterilization should be controlled. According to Molecular Cloning III, I set the autoclave 115℃ for 30 minutes, but I found the solution turned to brown-yellow after sterilization. Why~~~
Day 17. 2008/04/06
Today, I did only a colony PCR, used the 15 samples I swung yesterday. Nevertheless, no any amplified fragments…
In the afternoon, I spread the 8 bacteria mixture. When I centrifuged them, there was only a tiny white pellet on each bottom of Eppendorf tubes. It like that I did failed on last ligation, most of the bacteria was killed by Amp. However, I will screen the Amp-resistant colonies, so I had to spread the little liquid, with X-gal(70) and IPTG(16).
Day 18. 2008/04/07
All my yesterday re-collected bacteria got growing failed! Did the Amp was too toxic? I left the plates in the incubator so that they, which still alive but just not grew into colonies (if there were), could grow freely.
I was afraid that I failed to detect the bands, because I didn’t do the electrophoresis well, I re-PCR some samples of them. Nothing, however…
Then I got 2/3 of the target-contained two test-tubes of bacteria into 4 new tubes. I want to expand them. I also streaked the two strains on a plate—hoping to get some single colonies.
In the afternoon, I transferred single colonies once again, from the three plates:16 of rape II, 3 of radish I, and 4 of radish II. Later, I spread a few liquid. I will do colony PCR tomorrow, also the expanded two.
Day 19. 2008/04/08
For that I have got the recons of radish-AFP, now I need to screen some from rape. Nevertheless, I did a colony PCR, used the DNA templates from tube A3, A5, B3, B5, C3, which were samples from rape; at the same time, I also tried one of the samples I expanding inoculated yesterday, for testify whether I expanded the straits successfully.
While I was doing colony PCR, I tried to extracted plasmids of the expanded two straits.
At noon, the electrophoresis photo told me that I didn’t got my expected fragments beside the expanded one, and once again, I found only faint bands of plasmid…
Was it caused by the not-enough bacteria? The liquid LB medium in each tube all showed a clear yellow, just looked like the un-inoculated LB color. So that I had to swing the test tubes at 37℃ again. This time I only swung the rape tubes—I must get recons of rape! About 13:00, I swung them, and at 15:36, I found two of them turned cloudy, which showed there were bacteria grew! Ok, I got a little relief. After streaking the two bacteria liquid, I left lab complacently. The next is relied on my colony PCR of them!
What?! This noon I found some colonies on my plates, of which both the new X-gal spread and the more old X-gal spread, showed blue color! So that it must be that the former X-gal had lost its activity, and finally, I saw the beautiful and lovely blue of the colonies!
The Amp-free plates got a lot of colonies, contrary, there were almost not colonies on the Amp plates. There were nothing on the empty transferred plate; the pUC-18 plate got the full plate of tiny colonies. They all verified that my transformation was succeed; however, there are a few colonies on the radish and rape ligation plate, showed that most of the bacteria died on the Amp plates. Was there anything wrong in my last ligation? If it were, I would say that I got a deficient ligation time, which at least one hour but I only maintained 45 minuets at most… but even though it may be wrong in the ligation time, the un-ligated empty T-vectors should be transferred into cells, then show lots of colonies on the plates, just like the last time… well, the more I thought, the more I got puzzled.
At last, I make four new plate, with two Amp-free and two Amp added, each of them was covered by X-gal and IPTG (40:16). I spread all my remained bacteria liquid, including the liquid of this and the last time, onto the two Amp-free plates after 1.5 hours’ swing. On each Amp-added plates, I transferred 8×5 colonies from my last plates, one radish plate and one rape plate. I wanted to find some recons from the last plates. Then I transferred four single colonies from the new grown colonies, one blue from radish, two white from radish, and one white from rape, into four test tubes, then I swung them overnight for tomorrow’s PCR.
Ok, hoping to see recons, I also want to try endonuclease digesting earlier!
Day 16. 2008/04/05
This morning, I got out my plates. Now I got some new colonies which came from my second transformation. They are almost white, but it can’t instead of a succeed transformation, for some may be fake positive.
I did a colony PCR, used the bacteria liquid I swung yesterday.
I collected some colonies from the two Amp-free plates, transferred the colonies into 8 test tubes, radish tubes and 4 rape tubes. Then the PCR result showed that two of the four samples contained my target fragment, (electrophoresis photo: 080405-ColonyPCR.jpg). So that I see a beam of light toward the next step. Once again, I transferred another 12 (or 13?) single colonies into test tubes.
Ok, tomorrow, another batch of colony PCR!
Here I have a strange problem, which about plasmid extraction reagent. Solution I is a mixture of glucose, EDTA, and Tris-HCl, all the manual say that it should be autoclave sterilized. Glucose is unstable in high temperature, so its sterilization should be controlled. According to Molecular Cloning III, I set the autoclave 115℃ for 30 minutes, but I found the solution turned to brown-yellow after sterilization. Why~~~
Day 17. 2008/04/06
Today, I did only a colony PCR, used the 15 samples I swung yesterday. Nevertheless, no any amplified fragments…
In the afternoon, I spread the 8 bacteria mixture. When I centrifuged them, there was only a tiny white pellet on each bottom of Eppendorf tubes. It like that I did failed on last ligation, most of the bacteria was killed by Amp. However, I will screen the Amp-resistant colonies, so I had to spread the little liquid, with X-gal(70) and IPTG(16).
Day 18. 2008/04/07
All my yesterday re-collected bacteria got growing failed! Did the Amp was too toxic? I left the plates in the incubator so that they, which still alive but just not grew into colonies (if there were), could grow freely.
I was afraid that I failed to detect the bands, because I didn’t do the electrophoresis well, I re-PCR some samples of them. Nothing, however…
Then I got 2/3 of the target-contained two test-tubes of bacteria into 4 new tubes. I want to expand them. I also streaked the two strains on a plate—hoping to get some single colonies.
In the afternoon, I transferred single colonies once again, from the three plates:16 of rape II, 3 of radish I, and 4 of radish II. Later, I spread a few liquid. I will do colony PCR tomorrow, also the expanded two.
Day 19. 2008/04/08
For that I have got the recons of radish-AFP, now I need to screen some from rape. Nevertheless, I did a colony PCR, used the DNA templates from tube A3, A5, B3, B5, C3, which were samples from rape; at the same time, I also tried one of the samples I expanding inoculated yesterday, for testify whether I expanded the straits successfully.
While I was doing colony PCR, I tried to extracted plasmids of the expanded two straits.
At noon, the electrophoresis photo told me that I didn’t got my expected fragments beside the expanded one, and once again, I found only faint bands of plasmid…
Was it caused by the not-enough bacteria? The liquid LB medium in each tube all showed a clear yellow, just looked like the un-inoculated LB color. So that I had to swing the test tubes at 37℃ again. This time I only swung the rape tubes—I must get recons of rape! About 13:00, I swung them, and at 15:36, I found two of them turned cloudy, which showed there were bacteria grew! Ok, I got a little relief. After streaking the two bacteria liquid, I left lab complacently. The next is relied on my colony PCR of them!