SBB10Ntbk-DanielSedor: Difference between revisions
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[[Template:SBB-PCA | Gene Synthesis from Oligos (PCA method)]]<br> | [[Template:SBB-PCA | Gene Synthesis from Oligos (PCA method)]]<br> | ||
[[Template:SBB-Protocols_Zymo3 | Zymo Gel Purification]]<br> | [[Template:SBB-Protocols_Zymo3 | Zymo Gel Purification]]<br> | ||
[[Template:SBB-Protocols_Enz4 | Ligation of EcoRI/BamHI digests]] | [[Template:SBB-Protocols_Enz4 | Ligation of EcoRI/BamHI digests]]<br> | ||
[[Template:SBB-Protocols_Micro1 | Transformation by heat-shock]]<br> | |||
Revision as of 13:23, 17 February 2010
sbb10
Construction of sbb10
PCR DS001/CA1600 on Bca1587 5-1 (420bp, gp=A)
PCR CA1597/DS002 on Bca1587 5-6 (767bp, gp=B)
----------------------------
PCR DS001 and DS002 on A+B (1051 bp, EcoRI/BamHI)
Sub into pBjk2741 (EcoRI/BamHI, 2170+910, L)
Product is pBjk2741-sb10 {<SB100x!}
----------------------------
DS001 Forward oligo for cloning of <SB100x!
CCATAgaattcATGagatctGGTAAATCTAAAGAAATCTCTCAGGAC
CA1600 CGAAACGCAGACGAGCTTTTTTGTGACGGTTCTGGAGCAGCGGTTTTTT
PCA assembly of sleepingBeauty (Bca1587)
CA1597 ATGCTGGAAGAAACCGGTACCAAAGTTTCTATCTCTACCGTTAAACGTG
PCA assembly of sleepingBeauty (Bca1587)
DS002 Reverse oligo for cloning of <SB100x!
ctgatGGATCCttaGTATTTGGTAGCGTTACCTT
Part:
gatctGGTAAATCTAAAGAAATCTCTCAGGACCTGCGTAAACGTATCGTTGACCTGCACAAATCTGGTTCTTCTCTGGGTGCTATCTCTAAACGTCTGGCTGTTCCGCGTTCTTCTGTTCAGACCATCGTTCGTAAATACAAACA
CCACGGTACCACCCAGCCGTCTTACCGTTCTGGTCGTCGTCGTGTTCTGTCTCCGCGTGACGAACGTACCCTGGTTCGTAAAGTTCAGATCAACCCGCGTACCACCGCTAAAGACCTGGTTAAAATGCTGGAAGAAACCGGTACC
AAAGTTTCTATCTCTACCGTTAAACGTGTTCTGTACCGTCACAACCTGAAAGGTCACTCTGCTCGTAAAAAACCGCTGCTCCAGAACCGTCACAAAAAAGCTCGTCTGCGTTTCGCTACCGCTCACGGTGACAAAGACCGTACCT
TCTGGCGTAACGTTCTGTGGTCTGACGAAACCAAAATCGAACTGTTCGGTCACAACGACCACCGTTACGTTTGGCGTAAAAAAGGTGAAGCTTGCAAACCGAAAAACACCATCCCGACCGTTAAACACGGTGGTGGTTCTATCAT
GCTGTGGGGTTGCTTCGCTGCTGGTGGTACCGGTGCTCTGCACAAAATCGACGGTATCATGGACGCTGTTCAGTACGTTGACATCCTGAAACAGCACCTGAAAACCTCTGTTCGTAAACTGAAACTGGGTCGTAAATGGGTTTTC
CAGCACGACAACGACCCGAAACACACCTCTAAAGTTGTTGCTAAATGGCTGAAAGACAACAAAGTTAAAGTTCTGGAATGGCCGTCTCAGTCTCCGGACCTGAACCCGATCGAAAACCTGTGGGCTGAACTGAAAAAACGTGTTC
GTGCTCGTCGTCCGACCAACCTGACCCAGCTGCACCAGCTGTGCCAGGAAGAATGGGCTAAAATCCACCCGACCTACTGCGAAAAACTGGTTGAAGGTTACCCGAAACGTCTGACCCAGGTTAAACAGTTCAAAGGTAACGCTAC
CAAATACTAAG
Relevant Protocols
SOEing PCR
Cloning by PCR
Gene Synthesis from Oligos (PCA method)
Zymo Gel Purification
Ligation of EcoRI/BamHI digests
Transformation by heat-shock
