2199 pET30b-Ecoli-Muts
Cloning of 2199 pET30b-Ecoli-Muts
[edit]Purpose: Cloning of2199 pET30b-Ecoli-Muts==
Date: 2014-9-15---
Notebook:0
Page:
Clone ID:2199
Experimenters: shanyi
Results
[edit]CLONING
[edit]PCR from Escherichia coli str. K-12 substr. MG1655 genomic DNA
Do not amplify more than 20 cycles for the fist PCR
1st fragment
Ecoli-Muts-Nde1-F tacgacatatgagtgcaatagaaaatttc
2199-Ecoli-Muts-R2 (gaaacgctggctg caC atgccagtg) cactggcatGtgcagccagcgtttc
2nd fragment
2199-Ecoli-Muts-F2 ctggctg caC atgccagtgcgcgatac
Ecoli-Muts-Xho1-R (gatttatcgcttgaagagcctggtg ctcgag tagct)
agctactcgagcaccaggctcttcaagcgataaatc
join two fragments by PCR again Ecoli-Muts-Nde1-F+ Ecoli-Muts-Xho1-R
Digest with Nde1 + Xho1
Colony Screening
[edit]Date: 2014_9
Make miniprep, use Nde1 + Xho1 to find positive clone
Cloning
[edit]PCR from Escherichia coli str. K-12 substr. MG1655 genomic DNA
Oligos:
Ecoli-Muts-Nde1-F (5_14_2014, A05)
Sequence TACGACATATGAGTGCAATAGAAAATTTC
2199-Ecoli-Muts-R2 (9_8_2014 E09)
(gaaacgctggctg caC atgccagtg)
cactggcatGtgcagccagcgtttc
2nd fragment
2199-Ecoli-Muts-F2: (9_8_2014, E10 )
ctggctg caC atgccagtgcgcgatac
Ecoli-Muts-Xho1-R: (5_14_2014, A06 )
(gatttatcgcttgaagagcctggtg ctcgag tagct)
agctactcgagcaccaggctcttcaagcgataaatc
join two fragments by PCR again
Ecoli-Muts-Nde1-F
Ecoli-Muts-Xho1-R
clone into vector pET30b