Sequencing insert from Lambda gt10 vector- HELP!

Stuart Brown SBROWN at GAES.GRIFFIN.PEACHNET.EDU
Wed Sep 28 09:34:57 EST 1994


In article <36ambb$d00 at noc.usfca.edu>
padmmv00 at ac.usfca.edu (Mandalaparthy) writes:
 
>
> I am looking for help in sequencing my cuticle protein insert from lambda
>gt 10 vector. The cuticle protein gene is about 500 bp long. I am thinking
>of ordering the primers for gt10 from promega. The vector is about 41 kb.
>None of the faculty in our dept has tried direct sequencing from the
>vector so I  dont have a reliable protocol as such. My advisor wants me to
>try the non-radioactive silver-sequence technique ( kit from promega) If
>any of you have any ideas or experience in sequencing by this method or
>any other method , it would be of great help.
>
> Please reply either by e-mail to padmmv00.dons.ac at usfca.edu or post
>directly on the net.
>
> ps The silver sequence tech involves amplification by PCR and then
>sequencing. Any help with a protocol  to PCR the insert from the 41 kb gt
>10 vector will also be really helpful.
>
First, how large os your insert in the vector.  You said that your target
gene is 500 bp, but is that all, or have you cloned 10-20 kb of flanking
seq?  If your insert is large, then you had better remove the lambda arms
and sub clone your target fragment into a more manageable sized vector.
There are several flanking sites in the GT10 polylinker that remove the
lambda arms, so you can do some digests, run them out on a gel, blot and
probe with something that lights up your target gene, then sub-clone the
appopriate fragment.
 
 
If your lambda clone has a small insert, you can easily amplify the whole
thing using PCR primers that match the polylinker (T7, SP6, M13 sites etc).
If your insert is small enough (<800 pb) you might just use this PCR
product for direct seqencing using those same primers.  Otherwise, you
might use some gene specific primers, or restrction digest the PCR product
and subclone the frags with your gene.
 
 
 
Stuart Brown                       |   Plant Genetic Resources
                                   |   Georgia Experiment Station
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