Team:Alberta/ByteCreation

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Figure 2.  

How To Create a Byte In order to format a part as an AB or BA form Byte, the part first needs to be cloned in to pAB or pBA, respectively. 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. To create an AB Byte from pAB, the universal primers pAB_F and pAB_R are used. For the creation of a BA Byte from pBA, the primers pBA_F and pBA_R are used (Figure 3).

Figure 3. 

After amplification, treatment with USERTM 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 or BA form Byte (Figure 4).

Figure 4. <img src="http://2009.igem.org/wiki/images/c/c3/Alberta_byteconstruction.png" width="500">

<P>The protocol for amplifying and digesting AB and BA Bytes can be found in our lab section</a>.

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