Difference between revisions of "Part:BBa K187000"
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In order to format a part as an AB form Byte, the part first needs to be cloned in to pAB. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette. Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part (see parts <partinfo>BBa_K187365</partinfo> and <partinfo>BBa_K187366</partinfo>). After amplification, treatment with USER mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB Byte. | In order to format a part as an AB form Byte, the part first needs to be cloned in to pAB. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette. Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part (see parts <partinfo>BBa_K187365</partinfo> and <partinfo>BBa_K187366</partinfo>). After amplification, treatment with USER mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB Byte. | ||
| − | From this point the Bytes are ready to be used in the bead platform construct. For more information please see the BioBytes Standard RFC #47 and [http://2009.igem.org/Team:Alberta/Project/ByteCreation]. | + | From this point the Bytes are ready to be used in the bead platform construct. For more information please see the BioBytes Standard RFC #47 and [[http://2009.igem.org/Team:Alberta/Project/ByteCreation]]. |
Revision as of 00:34, 22 October 2009
pAB is one of two universal plasmids for the BioBytes standardized in vitro assembly method. The other universal plasmid, pBA, is entered as BBa_K187001. These plasmids were designed and constructed from pUC19 and contain the pMB1 high copy origin. Unique cassettes were designed containing PstI, XbaI, and primer annealing regions complementary to the ‘A’ and ‘B’ ends.
In order to format a part as an AB form Byte, the part first needs to be cloned in to pAB. This is done using a XbaI and PstI digest of both the insert and backbone, followed by ligation to place the part inside of the AB or BA cassette. Once the part is cloned, universal PCR primers containing deoxyuridine residues are used to amplify the part (see parts BBa_K187365 and BBa_K187366). After amplification, treatment with USER mix (available from New England Biolabs) creates a nucleotide gap at the position of the uracil by first excising the uracil base by Uracil DNA glycosylase and then cleaving the phosphodiester backbone at the apyrimidinic site via Endonuclease VIII. The resulting short oligonucleotides are then purified away from the PCR product to produce mature 12 base 3' overhangs of the AB Byte.
From this point the Bytes are ready to be used in the bead platform construct. For more information please see the BioBytes Standard RFC #47 and http://2009.igem.org/Team:Alberta/Project/ByteCreation.