SBB10Ntbk-DanielSedor: Difference between revisions
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===1 March 2010=== | ===1 March 2010=== | ||
Performed [[Template:SBB-Protocols_PCR3 | SOEing PCR]] protocol using sbb10A+sbb10B as a template and DS001 and DS002 as oligos. | Performed [[Template:SBB-Protocols_PCR3 | SOEing PCR]] protocol using sbb10A+sbb10B as a template and DS001 and DS002 as oligos. | ||
===8 March 2010=== | |||
Filler | |||
Revision as of 21:10, 8 March 2010
Construction Files
sbb10: Sleeping beauty (SB100x)
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
sbb39: Magnesium-repressed Promoter
Eco/Bam transfer pBjh1601CK-Bjh1380
Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-Bjh1380 {PmgtCB}
sbb40: Nuclear localization peptide
Eco/Bam transfer pBjh1601CK-Bjh1858
Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-Bjh1858 {<NIS!}
Relevant Protocols
Lab Notes
17 February 2010
Prepared two PCR's according to the Cloning by PCR protocol using oligos DS001/CA1600 (gp=A) and DS002/CA1597 (gp=B) from the sbb10 construction file.
Submitted preparation for thermocycling.
22 February 2010
Mixed 6μL of PCR products sbb10A and sbb10B (2/22 versions) with 4μL of Loading Buffer for the purposes of running a preparative gel (see SOEing by PCR protocol for more details).
Prepared two new PCR's according to the Cloning by PCR protocol.
24 February 2010
Mixed 6μL of PCR products sbb10A and sbb10B (2/22 versions) with 4μL of Loading Buffer for the purposes of running a preparative gel (see SOEing by PCR protocol for more details).
1 March 2010
Performed SOEing PCR protocol using sbb10A+sbb10B as a template and DS001 and DS002 as oligos.
8 March 2010
Filler
