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

2-26-2010

Additional lab time was provided today (Friday). I finished the zymo gel purification for the sbb09 part digest.

I also setup my final SOEing PCR reaction using the RH-33-A,B PCR products as template DNA.

2-24-2010

In order to debug the lack of band for sbb33-A from last lab session, I setup preparative gel samples for sbb33A 2k55, 2k45, 2k45 DSMO. No band appeared for 2k55. It appears that the annealing temperature was the reason no band showed up in the previous gel for this PCR product. Bands appeared for sbb33A when the 2k45 and 2k45 DMSO PCR protocols were used. I cut out the sbb33-A product from the gel, mixed it with the sbb33-B cut out, and performed a gel zymo purification on this mixture. I stored the result in a tube labeled "RH-clean-33-A and B". Preparative Gel for sbb33-A repeats

I took the zymo cleaned sbb9 PCR product and did a EIPCR BglII digest on it. I began the gel purification of this digest. I stored the melted gel sample in a tube with 600uL of ADB buffer. Gel for sbb09 digest purification

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). Analytical gel for sbb09

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). Preparative gel for sbb33-A and sbb33-B

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).