Somebody left my T4 polymerase, T7 polymerase and T4 ligase (all I bought from NEB, cost me almost USD300) on top of the refrigerator for more than 1 week.
Any chances it is still functioning? any way to assay the efficiency?
Thanks.
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3 replies to this topic
#1
Adrian K
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Posted 06 March 2011 - 11:15 AM
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#2
Ameya P
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Posted 06 March 2011 - 10:45 PM
Hey Adrian,
Sorry to hear that. I can assure you, it wasn't me
.
You could do a routine PCR with primers to see if it is still working. Some housekeeping gene amplification. or some internal controls. or some plasmid before the insert.
This comes to me as I am writing to you.
1. Any plasmid you might might have inserted an antibiotic gene cassette. PCR to check if the polymerase is fine
2. Digest.
3. Use Ligase, put in the cassette.
4. Confirm with PCR again...
5. Advantage: You have your own plate of some gene knock out + you know everything is working...
BTW, why weren't you in the lab for a week?
Sorry to hear that. I can assure you, it wasn't me
. You could do a routine PCR with primers to see if it is still working. Some housekeeping gene amplification. or some internal controls. or some plasmid before the insert.
This comes to me as I am writing to you.
1. Any plasmid you might might have inserted an antibiotic gene cassette. PCR to check if the polymerase is fine
2. Digest.
3. Use Ligase, put in the cassette.
4. Confirm with PCR again...
5. Advantage: You have your own plate of some gene knock out + you know everything is working...
BTW, why weren't you in the lab for a week?
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Replication of this art is strictly prohibited without express permission of the artist
#3
Adrian K
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Posted 07 March 2011 - 03:52 AM
Hey gt_ameya,
LoLx... I know it wasn't you...
Actually the T4 and T7 polymerase is mean for generation of LIC fragments, for my own private project. I will take your advice and try it anyway, I was thought they might be a short cut way so that I can avoid doing extra work..
I was in the lab the whole week, but I did not touch or look into the fridge as I'm doing something else, plus, the lab is very messy...
LoLx... I know it wasn't you...
Actually the T4 and T7 polymerase is mean for generation of LIC fragments, for my own private project. I will take your advice and try it anyway, I was thought they might be a short cut way so that I can avoid doing extra work..
I was in the lab the whole week, but I did not touch or look into the fridge as I'm doing something else, plus, the lab is very messy...
gt_ameya, on 06 March 2011 - 10:45 PM, said:
Hey Adrian,
Sorry to hear that. I can assure you, it wasn't me .
You could do a routine PCR with primers to see if it is still working. Some housekeeping gene amplification. or some internal controls. or some plasmid before the insert.
This comes to me as I am writing to you.
1. Any plasmid you might might have inserted an antibiotic gene cassette. PCR to check if the polymerase is fine
2. Digest.
3. Use Ligase, put in the cassette.
4. Confirm with PCR again...
5. Advantage: You have your own plate of some gene knock out + you know everything is working...
BTW, why weren't you in the lab for a week?
Sorry to hear that. I can assure you, it wasn't me
. You could do a routine PCR with primers to see if it is still working. Some housekeeping gene amplification. or some internal controls. or some plasmid before the insert.
This comes to me as I am writing to you.
1. Any plasmid you might might have inserted an antibiotic gene cassette. PCR to check if the polymerase is fine
2. Digest.
3. Use Ligase, put in the cassette.
4. Confirm with PCR again...
5. Advantage: You have your own plate of some gene knock out + you know everything is working...
BTW, why weren't you in the lab for a week?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#4
perneseblue
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Unlimited ligation works!
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Neutral
Posted 09 March 2011 - 10:20 PM
And check the T4 ligase as well. This enzyme denatures easily.
May your PCR products be long, your protocols short and your boss on holiday
