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From 2010.igem.org

Module I, group 2(b)

Change the plasmid for part I. pUC19 is suitable for most of the restriction sites and it is highly copied inside the E. coli. Thus primers 1-N-up (or do) have changed as follow.

eGFP

 1-E-up	5’-CAGCGTCGACTAGGGATAACAGGGTAATAGGAGGTCACTGTAATGGTGAGCAAGGGCGAG-3’(SalI)
 1-E-do	5’-CCACAAGCTTTTACTTGTACAGCTCGTCCATGCCG-3’(HindIII)

mCherry

 1-M-up	5’-CAGCGTCGACTAGGGATAACAGGGTAATAGGAGGTCACTGTAATGGTGAGCAAGGGCGAG-3’(SalI)
 1-M-do	5’-CCAC GGATCC CTACTTGTACAGCTCGTCCATGCCG-3’(BamHI)

Kan

 1-K-up	5’-CAGCGGATCCTAGGGATAACAGGGTAATCGGCATGGACGAGCTGTACAAGTAAGGAGGTCACTGATGATTGAACAAGATGGATTGCAC-3’(BamHI)
 1-K-do	5’-CCACGGTACCACGTTCGCTAATGCCGTTGACGGCCTCCCCTTATTAGAAGAACTCGTCAA-3’(KpnI)

Chlr

 1-C-up	5'-CAGCGGTACCTAGGGATAACAGGGTAATCGGCATGGACGAGCTGTACAAGTAAGGAGGTTGCTAAA ATGGAGAAAAAAATCACTG-3'(KpnI)
 1-C-do	5'-CCACGAATTCATTACCCTGTTATCCCTAACGTTCGCTAATGCCGTTGACGGCCCTCATCGCAGTACTGTTGTATTCAT-3'(EcoRI)

Find that the SuperMix is powerful in running PCR. It’s great news.

Module I, YX and ZY's part:

We PCR amplified the Kanamycin resistance and Chloramphenicol resistance genes. We also amplified the replication origin from pTKS/CS. Later, we ligated the PCR products into T vector.

We transformed competent trans5a cells with these T vectors and placed them on LB media with Kanamycin.

Module I, group 2c

We have tried various conditions for PCR, however, we failed to make any progress. To get the most suitable PCR reaction condition, we attempted to use Tm gradient and concentration gradients of template. 1/50,1/2500,1/125000 of previous template are used today. Besides, we tried 51℃, 53℃and 55℃ as Tm, respectively.

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Result: it seems that we can do nothing to adjust the condition. Success is so far from us!