Day 9. 2008/03/27
One of the two plates got all the three bacteria colonies, (and some of the colonies grew together, became to moss… oh, my, this time they grew too robust!)
■Chose some single colonies and transferred to liquid LB medium test-tubes, incubated at 37℃ by swing.
In the afternoon, I tried colony-PCR first time. I originally only wanted to test 15 samples (colonies), but, what’s wrong with me, I made a PCR mixture which enough to do 25 samples, (I think that I must have got jumbled)
So that I had to do 25 samples; the tiny tube tips made my finger pain! After putting the tubes into PCR apparatus, I clocked for 1.5 hours for prepare the gel. Everything seemed OK this time, but when I saw it about 1 hour later… my PCR had been stopped… who did it!? However, only God knows…
Day 10. 2008/03/28
In the morning I tried a raw-test for the incubated bacterium in test tubes.
However, I saw nothing but RNA fragments… so that I wanted to extract the plasmids in the afternoon.
I got some solution I, II, III from a classmate who also extracted plasmids then. This time when I added about 1ml bacteria liquid, I also added 0.5μl to each PCR reaction tube. This afternoon I passed in a full and busy tempo—extraction plasmids, amplifying my target fragment DNAs by PCR, before the end of PCR, I also made a gel for the later electrophoresis.
This afternoon I got nothing but RNA fragments, however…
I don’t know which step I got missed, the plasmids should be extracted well in theory…well, try again in the next week!
Day 11. 2008/03/31
■Add some fresh medium to each test tube to let them grow more, swing at 37℃ for some hours.
■Did another PCR by another four single colonies.
■Extracted the plasmids once again.
But still I got nothing but RNAs, neither my extracted plasmids or the PCR results…
Day 12. 2008/04/01
Why I always get nothing from my plasmid extraction? No matter of what the plasmid they are, at least I should see the empty vectors which survived on the Amp added plates. Well, this morning, I did it again, and this time, I subtracted the last step of ethanol washing. Then I detected by electrophoresis, I get some bands this time, however, they are faint, very faint… and me too… (see the mixed photo: 080401-plasmid_ps.jpg)
Another painful experience, of the blue-white screening, I got nothing showed blue, it’s impossible that I could get all the colonies absorbed the ligated vectors and then grew to recons. I doubted that it might be caused by I added not enough X-gal and IPTG… so that I made a mixture of X-gal(80μl) and IPTG(32μl), then spread half to two plates. After the liquid dried, I picked some single colonies, and transferred each to the two plates. I almost repeat the cycle 100 times: burn for sterilizing, cool it and pick out a single colony, transfer to another plate… not difficult but trouble…
Ok, I incubated the plates at 37℃, oh, colors, I want to see you!
Day 13. 2008/04/02
Maybe the X-gal got something wrong, yesterday’s plates still didn’t show any color.
My lab bought another type of competent cell, DH5α. This afternoon I used it to do another transformation. The reaction was the same like last time.
After transformation and one-hour swing, I added 65μl X-gal and 8μl IPTG into each tube. For I only had two plates then, I had to spread the four tubes of bacterium, (transformation of Radish, Rape, pUC18, and a empty competent cell), into the two plates, and each bacteria I got 85μl for spreading, (hoping it was ok -__-||).
Day 14. 2008/04/03
Hah! There was nothing on the plates when I came to lab this morning. Ok, E. coli, I’ll give you some fresh medium LB and swing you and spread the plates once again!
■This morning I prepared 5 new plates.
■At noon, I mixed X-gal and IPTG in proportion, (40μl:16μl), and this time, I changed to another new tube of X-gal. Then I spread the mixture of 56μl on the surface of my LB plates.
■Waiting for a hour, I got the re-swung tubes, centrifuged, discarded the supernatant and remained about 200μl of it; spread each plate by adding 120μl of bacteria liquid.
■When I put the plates into the incubator, surprisingly, the pUC-18 transferred part got a large of tiny colonies! But the other parts didn’t get, why?
■PS: I made four Amp-contained plates and one Amp-free plate, for to verify the “empty” transferred bacteria is still alive. I also spread the bacterium which I saved last time, (about 20μl in each tube).
■[COMMENT] I felt that during the two weeks, I still repeat the same work: ligation, transferring, and screening. I even didn’t budge little forward. It was not good, however: the extracted plasmids was too few to continue, the transferred cells grow not well, the blue-white screening was no any effect, and the worst is that I don’t know what’s the root of them. Expecting to get some steps upper …
One of the two plates got all the three bacteria colonies, (and some of the colonies grew together, became to moss… oh, my, this time they grew too robust!)
■Chose some single colonies and transferred to liquid LB medium test-tubes, incubated at 37℃ by swing.
In the afternoon, I tried colony-PCR first time. I originally only wanted to test 15 samples (colonies), but, what’s wrong with me, I made a PCR mixture which enough to do 25 samples, (I think that I must have got jumbled)
So that I had to do 25 samples; the tiny tube tips made my finger pain! After putting the tubes into PCR apparatus, I clocked for 1.5 hours for prepare the gel. Everything seemed OK this time, but when I saw it about 1 hour later… my PCR had been stopped… who did it!? However, only God knows…
Day 10. 2008/03/28
In the morning I tried a raw-test for the incubated bacterium in test tubes.
However, I saw nothing but RNA fragments… so that I wanted to extract the plasmids in the afternoon.
I got some solution I, II, III from a classmate who also extracted plasmids then. This time when I added about 1ml bacteria liquid, I also added 0.5μl to each PCR reaction tube. This afternoon I passed in a full and busy tempo—extraction plasmids, amplifying my target fragment DNAs by PCR, before the end of PCR, I also made a gel for the later electrophoresis.
This afternoon I got nothing but RNA fragments, however…
I don’t know which step I got missed, the plasmids should be extracted well in theory…well, try again in the next week!
Day 11. 2008/03/31
■Add some fresh medium to each test tube to let them grow more, swing at 37℃ for some hours.
■Did another PCR by another four single colonies.
■Extracted the plasmids once again.
But still I got nothing but RNAs, neither my extracted plasmids or the PCR results…
Day 12. 2008/04/01
Why I always get nothing from my plasmid extraction? No matter of what the plasmid they are, at least I should see the empty vectors which survived on the Amp added plates. Well, this morning, I did it again, and this time, I subtracted the last step of ethanol washing. Then I detected by electrophoresis, I get some bands this time, however, they are faint, very faint… and me too… (see the mixed photo: 080401-plasmid_ps.jpg)
Another painful experience, of the blue-white screening, I got nothing showed blue, it’s impossible that I could get all the colonies absorbed the ligated vectors and then grew to recons. I doubted that it might be caused by I added not enough X-gal and IPTG… so that I made a mixture of X-gal(80μl) and IPTG(32μl), then spread half to two plates. After the liquid dried, I picked some single colonies, and transferred each to the two plates. I almost repeat the cycle 100 times: burn for sterilizing, cool it and pick out a single colony, transfer to another plate… not difficult but trouble…
Ok, I incubated the plates at 37℃, oh, colors, I want to see you!
Day 13. 2008/04/02
Maybe the X-gal got something wrong, yesterday’s plates still didn’t show any color.
My lab bought another type of competent cell, DH5α. This afternoon I used it to do another transformation. The reaction was the same like last time.
After transformation and one-hour swing, I added 65μl X-gal and 8μl IPTG into each tube. For I only had two plates then, I had to spread the four tubes of bacterium, (transformation of Radish, Rape, pUC18, and a empty competent cell), into the two plates, and each bacteria I got 85μl for spreading, (hoping it was ok -__-||).
Day 14. 2008/04/03
Hah! There was nothing on the plates when I came to lab this morning. Ok, E. coli, I’ll give you some fresh medium LB and swing you and spread the plates once again!
■This morning I prepared 5 new plates.
■At noon, I mixed X-gal and IPTG in proportion, (40μl:16μl), and this time, I changed to another new tube of X-gal. Then I spread the mixture of 56μl on the surface of my LB plates.
■Waiting for a hour, I got the re-swung tubes, centrifuged, discarded the supernatant and remained about 200μl of it; spread each plate by adding 120μl of bacteria liquid.
■When I put the plates into the incubator, surprisingly, the pUC-18 transferred part got a large of tiny colonies! But the other parts didn’t get, why?
■PS: I made four Amp-contained plates and one Amp-free plate, for to verify the “empty” transferred bacteria is still alive. I also spread the bacterium which I saved last time, (about 20μl in each tube).
■[COMMENT] I felt that during the two weeks, I still repeat the same work: ligation, transferring, and screening. I even didn’t budge little forward. It was not good, however: the extracted plasmids was too few to continue, the transferred cells grow not well, the blue-white screening was no any effect, and the worst is that I don’t know what’s the root of them. Expecting to get some steps upper …