SBB10Ntbk-RaffiHagopian
Construction files for the parts I'm making:
Construction of Tn5 3'TR (sbb09) BglBrick Part
EIPCR rh01F/rh02R on pBjk2741-Bca1144 (2230 bp, BglII) Product is pBjk2741-sbb09 {Tn5 3'TR} ---- rh01F EIPCR construction of Tn5 3'TR basic part ccataAGATCTggttgagatgtgtataagagacagtcgacGGATCCtaactcgctcctcag rh02R Reverse BglII oligo for pBjk2741 EIPCR ccaatAGATCTcatGAATTCcacttcag
JCA Notes
- correct
- similarity to other parts, changed construction:
Construction of Tn5 3'TR (sbb09) BglBrick Part
EIPCR rh01F/tn0002R on pBjk2741-Bca1144 (2230 bp, BglII) Product is pBjk2741-sbb09 {Tn5 3'TR} ---- rh01F EIPCR construction of Tn5 3'TR basic part ccataAGATCTggttgagatgtgtataagagacagtcgacGGATCCtaactcgctcctcag tn0002R Reverse BglII oligo for Tn5 EIPCR CCAATAGATCTcatgaattcCACTTCAG
Construction of Gentamicin resistance gene (sbb33) BglBrick Part PCR rh03F/rh05R on pLoxGen4 (576 bp, gp = A) PCR rh04F/rh06R on pLoxGen4 (284 bp, gp = B) ---- PCR rh03F/rh06R on A+B (bp 833, EcoRI/BamHI) Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 2170+910, L) Product is pBjk2741-sbb33 {Promoter.rbs.GenR} ---- rh03F Forward EcoRI for BglBricking GenR ctctgGAATTCatgAGATCTctagcgcgtcgacataagcc rh04F Removing the BglII site from GenR cgcgtagtgagatAtatatctatgatc rh05R Removing the BglII site from GenR gatcatagatataTatctcactacgcg rh06R Reverse BamHI for BglBricking GenR gcaaaGGATCCctagcgcgtcggccgggaag
JCA Notes
- correct
Since part isn't genR, replacing with new part:
PCR ca1749/rh05R on pG80 (621bp, frag 1) PCR rh04F/ca1750 on pG80 (254bp, frag 2) --------------------------------- PCR ca1749/ca1750 on 1+2 (848bp, Eco/Bam) Sub into pBjk2741 --------------------------------- Product is pBjk2741-sbb44 {Promoter.rbs.genR} --------------------------------- ca1749 Forward cloning of genR from pG80 cagtaGAATTCatgAGATCTCTGGCAGTTCCCTACTCTCG ca1750 Reverse cloning of genR from pG80 ccataGGATCCCTTGAACGAATTGTTAGGTGG rh04F Removing the BglII site from GenR cgcgtagtgagatAtatatctatgatc rh05R Removing the BglII site from GenR gatcatagatataTatctcactacgcg
Raffi Hagopian Tn5 3'TR (sbb09)
Raffi Hagopian Gentamicin resistance gene (sbb33)
2-22-2010
We received our PCR products today. I prepared two preparative gel samples for RH-PCR-33-A and B. I also prepared an analytical gel sample for RH-PCR-9. I performed a Regular Zymo cleanup on RH-PCR-9. I stored the clean DNA from this zymo procedure into a tube labeled "RH-9-Clean-PCR" (33uL). The "RH-9-Clean-PCR" tube was placed in "Box 140L C".
The analytical gel for RH-PCR-9 produced a band at the expected size (2230bp). On the preparative gel, RH-PCR-33B produced a band at the expected size (284bp). This band was sliced out of the gel and stored in box "C" in a tube labeled RH-GP-B.
RH-PCR-33A failed to produce a band. To debug this issue, I set up three PCR reactions for the sbb33A product (2k55, 2k45, 2k45+DMSO).
2-17-2010
Today was the first day of wet lab work. I made 100 uM stock solutions for oligos: rh01F,rh03F,rh04F,rh05R,rh06R. I obtained oligo tn0002R from a lab mate. I followed the Cloning by PCR protocol to setup the EIPCR mixture for part sbb09. I followed this same protocol another two times to set up the initial two SOEing PCR mixtures for part sbb33.
At the end of the day I had three tubes ready for PCR:
PCR Tube Label: RH-PCR-9 Description: EIPCR mixture for constructing part sbb09. Program: 4K55 PCR program (it's 2200bp long). PCR Tube Label: RH-PCR-33-A Description: The PCR mixture for obtaining SOEing product A for part sbb33. Program: 2K55 PCR program (it's under 2000bp long). PCR Tube Label: RH-PCR-33-B Description: The PCR mixture for obtaining SOEing product B for part sbb33. Program: 2K55 PCR program (it's under 2000bp long).