Team:Washington/Notebook/NheI

Gene Assembly using the NheI site

 * 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 58C
 * 5) Next start with the last 23 coding nucleotides eg.5'-tattttcagggtgctagctaa-3'
 * 6) Remove the stop codon(s), in this case taa, and replace with the SpeI cut site ACTAGT, eg. 5'-tattttcagggtgctagcACTAGT-3'
 * 7) Add 6-8 random nucleotides to the 3' end of the primer, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3'
 * 8) REVERSE COMPLEMENT PRIMER, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3'  --> 5'-gacccagACTAGTgctagcaccctgaaaata-3'
 * 9) Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
 * 10) Amplify your gene using the designed forward and reverse primers
 * 11) PCR purify the PCR product
 * 12) To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
 * 13) Digest PCR product with XbaI and SpeI
 * 14) PCR Purify
 * 15) Digest Vector with NheI and CIP
 * 16) PCR purify
 * 17) Mix insert and vector in 3:1 ratio and ligate
 * 18) Transform into competent cells
 * 19) SCREEN CELLS FOR CORRECT INSERT ORIENTATION by colony PCR using VF2 and custom reverse oligo