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ahmadfathi

Member Since 06 Feb 2013
Offline Last Active Apr 29 2013 05:33 PM

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cloning<br />protein expression

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2xzwei
04 Apr 2013 - 00:43

Topics I've Started

problem with sequencing and mapping

27 April 2013 - 06:07 PM

hello, right now, i already finished transform and co-transform my recombinant dna with supporters plasmid. the problem is when i doing sequencing, there an insertion of nucleotide occurred. this is impossible because my recombinant dna is a wild type dna..any suggestion or idea to solve this problem?

right nnow, i just redo sequencing cycle.

last one, what the software or website to generate the recombinant dna map? i used once and totally forgot..

Co-transformation

09 March 2013 - 02:43 AM

hello, i already done the transformation of my insert into new vector in dh5a host and extracted into a plasmid (CYP3A4-pcWori-dh5a plasmid). then my lecturer said that i need to perform a co-transformation in oxy-reductase vector (pCYAC-OXR) before express the protein, so can i just do the co-transformation of CYP3A4-pcWori-dh5a (Amp resistant) and pCYAC-OXR ( chloramphenicol resistant) plasmid like doing standard transformation? or there have different methods.
now, i just do the standard transformation by put in 25ng of CYP3A4-pcWori-dh5a+ 25ng pCYAC-OXR into dh5a competent cell and streak on chloramphenicol 100ug/ul plate..the result is negative.

PCR and restriction enzyme digestion

16 February 2013 - 03:45 AM

hello, i done transformation on dh5-alpha culture and the culture is grown, when i screen the culture by performing PCR, it give positive result (band shown at expected size). however, when i retest with double digestion, there no band even for the positive control. i used 200 ng and 10U (0.5 ul) at 37^oC for an hour then inactivate immediately at 65^oC for 20 minute? is it my transformation is really worked? any recommendation?

DNA ligation and trasformation

06 February 2013 - 06:50 PM

hello, i have performed ligation for my sample. the concentration of my vector is 8 ng/ul while insert is 15 ng/ul. i used 1:3 ratio of vector insert where insert + vector= 100ng..this sample done in 4,16,23 and 25^oC. The ligation product then stored in -20^oC and 4 ul of ligation product then used for transformation on dh5a. however i got no cloning culture. i used invitrogen t4 ligase..is there any problems in my methods?

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