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Old 10-21-2010, 10:41 AM   #1
manishbudathoki
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Default restriction endonuclease detection

5-GTGACCGCGGATATCGAATTCGGTACCATGAGTAAAGGAGAAGAACT AACTATACAAATAA-3

Which restriction endonucleases can theoretically be used for above cDNA subcloning?

how to Verify that the nucleotide sequence labeled in green codes for GFP (accession number AAA27722)


can you please explain the steps as well?
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Old 10-21-2010, 10:51 AM   #2
krobison
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1) NEBCutter
2) BLAST
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Old 10-21-2010, 01:02 PM   #3
malachig
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Vector NTI is a classic (although unfortunately now commercial) solution for dealing with cloning, restriction enzyme digestion, etc. Using this software you can load up a vector map for your vector, which will contain annotations of things like the GFP element.

As krobinson suggests you can use BLAST to confirm the likely identity of your pasted sequence. There are several possible strategies. For example:

1.) BLAST against all of nr using nucleotide blast

2.) Use the vector screening option of BLAST

3.) Download the expected matching sequence using your accession AAA27722 and then use a pairwise blast to compare your sequence. Make sure to select the 'Somewhat similar sequences (blastn)' option. This will produce the attached result:
Attached Files
File Type: txt C16VXDR2112-Alignment.txt (1,005 Bytes, 3 views)
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