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'
   1. Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
9. Amplify your gene using the designed forward and reverse primers
10. PCR purify the PCR product
    1. To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
11. Digest PCR product with XbaI and SpeI
    1. PCR Purify
12. Digest Vector with NheI and CIP
    1. PCR purify
13. Mix insert and vector in 3:1 ratio and ligate
14. Transform into competent cells
15. SCREEN CELLS FOR CORRECT INSERT ORIENTATION by colony PCR using VF2 and custom reverse oligo