"

From 2010.igem.org

We have got a new office room: Room 101,Old Biology Building.

Module I, group 2(b)

Change the protocol again. We now have two idea of ligation the four fragments. One is linking the fragments one by one into the plasmid pTKIP, with modified primers fit the sequence of the restriction sites along the plasmid. Another is using the DraIII site (CACNNNGTG, N stands for any nucleotide) to design multiple sites which cannot be ligated oneself. The newly primers’ sequences and ID numbers are shown below.

eGFP

 1-E-up	5’-CAGCGTCGACTAGGGATAACAGGGTAATAGGAGGTCACTGTAATGGTGAGCAAGGGCGAG-3’(SalI)
 1-E-do	5’-CCAC AAGCTT TTACTTGTACAGCTCGTCCATGCCG-3’(HindIII)
 2-E-up	5’-CAGCGTCGACTAGGGATAACAGGGTAATAGGAGGTCACTGTAATGGTGAGCAAGGGCGAG-3’(SalI)
 2-E-do	5’-CCACCACGGAGTGTTACTTGTACAGCTCGTCCATGC-3’(DraIII)

mCherry

 1-M-up	5’-CAGCAAGCTTTAGGGATAACAGGGTAATAGGAGGTCACTGTAATGGTGAGCAAGGGCGAG-3’(HindIII)
 1-M-do	5’-CGCGAATTCATTACCCTGTTATCCCTACTACTTGTACAGCTCGTCCATGC-3’(EcoRI)
 2-M-up	5’-CAGCCACTCCGTGTAGGGATAACAGGGTAATAGG-3’(DraIII)
 2-M-do	5’-CCACCACCTGGTGCTACTTGTACAGCTCGTCCATG-3’(DraIII)

Kan

 1-K-up	5’-CAGCGGGCCCTAGGGATAACAGGGTAATCG-3’(ApaI)
 1-K-do	5’-CCACGTCGACACGTTCGCTAATGCCGT-3’(SalI)
 2-K-up	5’-CAGCCACCAGGTGTAGGGATAACAGGGTAATCGG-3’(DraIII)
 2-K-do	5’-CCACCACCCGGTGACGTTCGCTAATGCCGTT-3’(DraIII)

Chlr

 1-C-up	5'-CAGCGGTACCTAGGGATAACAGGGTAATCGGCATGGACGAGCTGTACAAGTAAGGAGGTTGCTAAA ATGGAGAAAAAAATCACTG-3'(KpnI)
 1-C-do	5'-CCACGGGCCCACGTTCGCTAATGCCGTTG-3'(ApaI)
 2-C-up	5'-CAGCCACCGGGTGTAGGGATAACAGGGTAATCGG-3'(DraIII)
 2-C-do	5'-CCACGAATTCATTACCCTGTTATCCCTAACGTTCGCTAATGCCGTTGACGGCCCTCATCGCAGTACTGTTGTATTCAT-3'(EcoRI)