PCR a fragment of 6kb - (Jun/26/2008 )
QUOTE (ph3no @ Jul 4 2008, 09:50 AM)
and since you were talking about being able to clone 3kb frags, I assume you are electroporating as well.
I've gone up to 15 kb with heath-shock. (total vector size after ligation this was, not 15 kb PCR in a topo-TA-vector).
-vairus-
QUOTE (ph3no @ Jul 4 2008, 09:50 AM)
QUOTE (jiro_killua @ Jun 27 2008, 09:08 AM)
1. template is mouse kidney cDNA (TRIZol, then superscript 3)
2. agree, the thing is I am using TA cloning, should be easy, right? But I keep failing to get positive clones when I PCR check clones, so I wonder if imcreasing the concentration of the PCR product (but not the absolute amount of PCR product) will help
2. agree, the thing is I am using TA cloning, should be easy, right? But I keep failing to get positive clones when I PCR check clones, so I wonder if imcreasing the concentration of the PCR product (but not the absolute amount of PCR product) will help
wooooow... I agree with Phusion, it's the best choice. But if you cannot clone a 6Kb fragment into a 3.5Kb vector... I assume that you are using invitrogen Topo TA cloning kit or similar... thus the vector is kinda small as compared with your insert hence the absence of positive clones... but I might be wrong... look at this link :
http://www.clontech.com/images/ctq/APR07UP...epPCR_IF_US.pdf
I'd give clonetech InFusion a go...
and since you were talking about being able to clone 3kb frags, I assume you are electroporating as well.
Yes, I am using Invitrogen TA cloning kit (pCR2.1) with a vector size of 3.9kb
I do know that insert size will affect ligation efficiency, but I don't know there is a limit (well, of course there should be, I assume you cannot ligate something 100kb into a 4kb vector).
Is it a problem with 6kb insert into 4kb vector?
-jiro_killua-
Just want to thanks you guys!!!
Phusion seem to work very well
the PCR product is at least 100 times brighter than using Platinum HiFi Taq
And I got colonies after TA cloning
Just need to wait and see if I have any mutation in that 6kb gene
-jiro_killua-
congratulations 
,
what conditions did you now use?
-Ned Land-
QUOTE (Ned Land @ Jul 21 2008, 09:35 AM)
congratulations ,
what conditions did you now use?
,what conditions did you now use?
Enzyme: Phusion
Buffer: 5X HF buffer
additional MgCl2 not required
Thermal cycle:
98C 20s
98C 20s
60C 10s
72C 2m30s
(30 cycles)
72C 10m
Then add Taq, incubate for 10min at 72C
Then either purify with column, or directly use for TA cloning
I sequenced the first 900kb, no mutation, yet
I'll keep my fingers crossed
-jiro_killua-