Team:Washington/Notebook/NheI PstI

Gene Assembly using the NheI and PstI sites

 * 1) Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
 * 2) Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
 * 3) Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
 * 4) Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to that of VR
 * 5) Amplify your gene using the designed forward oligo and VR
 * 6) PCR purify the PCR product
 * 7) To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
 * 8) Digest PCR product with XbaI and PstI
 * 9) PCR purify
 * 10) Digest Vector with NheI and PstI
 * 11) PCR purify
 * 12) Mix insert and vector in 3:1 ratio and ligate
 * 13) Transform into competent cells
 * 14) Screen cells for correct insert using VF2 and VR