SBB10Ntbk-RaffiHagopian

Construction files for the parts I'm making:

  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

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).