Dear All,
I am thinking if I was given a vector pTZ57R, and have to turn it into TA cloning pTZ57R/T (Fermentas vector) vector,
I have to use EcoRV (Eco32I) to cut it. However, EcoRV is a blunt end cutter:
5'...GAT^TAG...3'
3'...CTA^ATC...5'
My question is, how can we make a "ddT" after "GAT" and another "ddT" before "ATC" ( refer to the Fermentas InsTAclone manual, between 650 and 651 of MCS region)
I know we can make "a-overhang" in PCR fragment by incubate with Taq polymerase but i didn't know how to add "T-overhang"
Any idea?
InsTAclone PCR Cloning Kit #K1213, #K1214
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2 replies to this topic
#1
Adrian K
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Posted 30 March 2010 - 09:44 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
rkay447
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Posted 30 March 2010 - 02:48 PM
There are protocols on this website for T-vector construction:
http://www.protocol-...ctor/index.html
http://www.protocol-...ctor/index.html
#3
Adrian K
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Posted 30 March 2010 - 05:38 PM
Oh thanks for the reminder....I didn't notice that...
Thanks again.
Thanks again.
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
