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Construction file for sbb19

 Construction of FokI Cleavage Domain basic part sbb19
 PCR fk0001/fk0003 on BBa_K243000 (504 bp, gp = A)
 PCR fk0004/mb0002 on BBa_K243000 (144 bp, gp = B)
 PCR fk0001/mb0002 on A+B           (622 bp, EcoRI/BamHI)
 Digest pBjk2741-Bca1144          (EcoRI/BamHI, 2170+910, L)
 Product is pBjk2741-sbb19	      {<FokI+!}
fk0001   Forward EcoRI and BglII for FokI 	 
fk0004   (F)Base Substitution FokI   
fk0003   (R)Base Substitution FokI   
mb0002  Reverse BamHI  

Construction file for sbb23

 Construction of Zinc Finger Target basic part sbb23
 Wobble mb0005/mb0006           (68bp, EcoRI/BamHI)
 Sub into pBca9145-Bca1144#5    (EcoRI/BamHI, 2057+910, L)
 Product is pBca9145-sbb23      {ZFNS target}
 mb0005   Forward construction of ZFNS target basic part
 mb0006   Reverse construction of ZFNS target basic part

14:43, 22 February 2010

  • Preparative gel on PCR_A and PCR_B for sbb19, zymo gel purification on A+B, eluted into 50 uL water, labelled "MB SBB19 A+B Temp", put into box C
  • Small frag zymo cleanup on sbb23 wobble product, labelled "MB SBB23", put into box C

12:18, 24 February 2010

Plan: *Complete SOEing of sbb19 by pcr-ing external oligos, using pcr products A and B (in the same tube) as template
*Digest wobble product for sbb23, gp, ligation, transformation

Cloning by PCR
EcoRI/BamHI Digest of Wobble Products
Zymo Gel Purification
Ligation of EcoRI/BamHI digests
Transformation by heat-shock

  • Prepared final PCR for SOEing of sbb19, using oligos fk0001 and mb0002 on template A+B, analytical gel on product confirmed correct
  • Digested wobble product for sbb23 with EcoRI/BamHI, incubated 1 hr @37 Celcius, preparative gel on product confirmed correct, melted gel with 600 uL ADB buffer and labelled "MB SBB23 prep", put into box C

12:38, 1 March 2010

Plan: *zymo the completed SOE product for sbb19
*Digest SOE product for sbb19 with EcoRI/BamHI, gp
*Finish zymo gp, ligate digested, gp'd wobble product for sbb23 into pBca9145-Bca1144#5
*Transform for sbb23 if time

Regular Zymo Cleanup

  • Zymo'd the completed SOEing pcr product for sbb19, labelled "MB sbb19 A+B Pure", put into box C.
  • Digested a portion of purified sbb19 SOE pcr product with E/Ba using standard procedure, melted gel into 600 uL ADB, labelled "MB sbb19 PURE DI", put into box C
  • Finished zymo gp of sbb23 wobble digest, eluted into 33 uL water, labelled "MB sbb23 wobl PURE DI", put into box C

12:14, 3 March 2010

Plan: *Ligate sbb23 wobble digest "MB sbb23 wobl PURE DI" with pBca9145, wait the 30 minutes
*While waiting, finish zymo of sbb19 digest "MB sbb19 PURE DI" (bad name, isn't pure yet)
*Ligate sbb19 digest with pBjk2741, wait the 30 minutes
*Transformation for both sbb19 and sbb23

Ligation of EcoRI/BamHI digests
Transformation by heat-shock

  • Added 17 uL of water to "MB sbb23 wobl PURE DI", now 50 uL total (was 33 uL which was an incorrect concentration, concentration coming out should be same as that used at the start)
  • Ligated MB sbb23 wobl PURE DI with pBca9145
  • Finished zymo of sbb 19 digest, eluted into 8 uL water to match the concentration at the start of the digestion process, labelled "MB sbb19 PURE DI", put into box C
  • Aborted incorrect transform that used LB media instead of KCM
  • Re-did ligation of "MB sbb 23 wobl PURE DI" with pBca9145
  • Transform for sbb23, plating done by Will or Mike (Thanks)

12:13, 8 March 2010

Plan:  *Ligate sbb19 SOEing digest "MB sbb19 PURE DI" with pBjk2741
*While waiting, Machery-Nagel miniprep on sbb23-transformed cells
*Transform sbb19-vector complex

Macherey-Nagel Miniprep

  • Ligated sbb19 digest with pBjk2741, incubated on benchtop 30 min
  • Started M-N miniprep for sbb23, colony 2 was red (parent vector) so discarded.
  • Transformation for sbb19, incubated @37 deg C 1 hr
  • Finished M-N miniprep for sbb23 while waiting, labelled tubes "MB sbb23-1 MiniP" etc, put into box C
  • Plated sbb19 on spec (ab)

12:22, 10 March 2010

 Plan: *Analytical digest on sbb23 M-N minipreps 1, 3, and 4.
*Begin M-N miniprep of sbb19-transformed cells
*Analytical gel on sbb23 digests
*Finish M-N miniprep of sbb19-transformed cells

Analytical digests (Mapping)

  • Analytic digest of sbb23-1 MiniP etc
  • M-N miniprep of sbb19-transformed cells, labelled "MB sbb19-1 MiniP" etc, put into box C. Note that the LB used for rescue was contaminated, and the plates were covered in little white dots.
  • Realized that I used BamHI instead of XhoI (for small parts) in the sbb23 analytical digest, so redid. Not really necessary since bam and xho are right next to each other in pBca9145
  • Analytical gel on sbb23 miniprep digests, fragment lengths should be 61 BP and 2048 BP

Mb031010.jpgladder - sbb23 clone 1 - sbb23 clone 3 - sbb23 clone 4

Clone 1 vector band is slightly displaced from that of clones 3 and 4. This does not imply that clone 1 is bad, and may in fact mean quite the opposite. The part band is not visible in any of the clones due to its extremely small size and the low DNA concentration relative to post-PCR gels.

1:48, 15 March 2010

  • Analytical digest of sbb19-1 MiniP etc, analytical gel. Had to restain with EtBR for ~10 min.

Mb 03 15 10.jpg
ladder - sbb19 clone1 - sbb19 clone2 - sbb19 clone3 - sbb19 clone4

All of my clones look good, with bands near 622 and 2170. Sent in sbb19 clones 1 and 2 for sequencing. Sent in sbb23 clones 3 and 4 for sequencing.

2:06 March 17, 2010

  • sbb19 sequencing looks perfect (well E4)
  • sbb23 sequencing shows up as parent vector, but analytical gel with EcoRI/XhoI should have exposed this with an RFP band. Remapping with AlwNI and BglII. If the part is correct, two bands should be visible (1516 and 593). If not, only one band should be visible (~1500)

19:45 May 5, 2010

See Team 1: Toxicity and Expression for all information regarding all assays.