=cloning problems

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Tue Aug 20 10:04:16 EST 2002


Make sure that when you visualize the fragments by UV, that they are 
not overexposed.  A long-wave hand held UV lamp is what I use for 
visualizing fragments I am cutting out of gels-- this causes little 
if any damage to fragments.  However, many UV light boxes can destroy 
fragments for cloning quite rapidly.

Mike

>Hi, my friends,
>   I have sent this message once before.But till now I still cannot make it.
>   Please help me.
>   I'm in great trouble with my contruction of expression plasmids.The
>vectors and fragments were all double digested by the same two 
>enzymes and target bands after electrophoresis were extracted out 
>from gel by the gel-extraction kit
>from Roche or by freeze-thraw method.The extraction was very good 
>checked by selectrophoresis.For ligation,I tried many times and
>many ways including rapid ligation kits from different companies and 
>individual reagents.I can't make it always.I am so discouraged.
>     Would anyone give me some suggestion and and information for my
>difficulty.
>     Thanks in advance.
>
>     Huazhong
>
>
>
>
>
>   Wang Hua-zhong  Ph.D
>   Cytogenetics Institute,
>   Department of Agronomy ,
>   Nanjing Agricultural University,
>   Nanjing,Jiangsu,P.R.China
>   210095

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