Side Project 1--Toggle Switch



       Objective 1: Determine if we can use heterochromatin to build a functional toggle switch with memory

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 Objective 1: Determine if we can use heterochromatin to build a functional toggle switch with memory.

Milestones:

<p class=listparagraph style='text-indent:-.25in'>A)<span style='font-size: 7.0pt;font-family:"Times New Roman"'>    Obtain all the parts needed to build the toggle switch.

<p class=listparagraph style='text-indent:-.25in'>B)<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Build toggle switch using the parts.

<p class=listparagraph style='text-indent:-.25in'>C)<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Integrate toggle switch into a yeast strain with LexA-SIR2.

<p class=MsoNormal>Rationale: Demonstrate the modularity of the chromatin bit by building a toggle switch using chromatin.

<p class=MsoNormal>Steps needed to accomplish objective.

<p class=listparagraph style='text-indent:-.25in'>1.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     PCR parts from …….

<p class=listparagraph style='margin-left:1.0in;text-indent:-.25in'>a.<span style='font-size:7.0pt;font-family:"Times New Roman"'> Topo Clone.

<p class=listparagraph style='margin-left:1.0in;text-indent:-.25in'>b.<span style='font-size:7.0pt;font-family:"Times New Roman"'> Sequence Topo Clone

<p class=listparagraph style='text-indent:-.25in'>2.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Ligate parts into PAH32 and PAH77.

<p class=listparagraph style='text-indent:-.25in'>3.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Integrate GAL1P*/ADH1P*LexA-SIR2-mCherry and LexAop-ADH1P-TetR-yEGFP into a yeast strain (SV992).

<p class=listparagraph style='text-indent:-.25in'>4.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Test toggle switch using FACS.

<p class=listparagraph style='text-indent:-.25in'>5.<span style='font-size:7.0pt;font-family:"Times New Roman"'><i> </i></b> ' GIVE PROJECT TO LINGLI/TIFFANY. '

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 1 (7/7/08 &#8211; 7/13/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Discuss with Andrew about the project.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- PCR’ed up using ADH1P* and GAL1* plasmid from Collins paper as the templates.

<ul style='margin-top:0in' type=disc> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt;mso-list: l0 level1 lfo2;tab-stops:list .5in'>Parts:</li> <ul style='margin-top:0in' type=circle> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>TetR with Xhol/Bamh1 ends.</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>TetR with AB ends for AaR1 digest.</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>yEGFP with XhoI/BamhI ends.</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>yEGFP with BamhI/NotI ends.</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>Gal10/1* with PspomI/XhoI ends.</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>Adh1p* with PspomI/XhoI ends.</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l0 level2 lfo2;tab-stops:list 1.0in'>Cyc1t with NotI/Sac1 ends.</li> </ul> </ul>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Dpn1 treated the PCR reaction.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned failed. There were 0 to 3 colonies on each plate.

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:.25in;margin-bottom:.0001pt'>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 2 (7/14/08 &#8211; 7/20/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Gel purified PCR product from last week PCR reaction.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned again.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned failed again. Zero colonies on the plates. The reason why Topo clone failed could’ve been that I didn’t dry the X-gal +IPTG plate before plating the cells.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned again using the same PCR product. This time the plates are dried before cells are plated onto.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Digest PAH32 with XhoI &amp; Bamh1, PAH32 with Xho1 &amp; NotI, and AH81 AlexA with PspomI &amp; XhoI.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Gel purified vector PAH32, PAH32 and AH81 AlexA.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned failed again. Zero colonies on the plates.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- PCR’ed toggle switch parts again using the same protocol.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- I saved 4.5ul of the PCR reaction and Dpn1 treated the rest of the reaction.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned both Dpn1 treated and not treated.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo clone is successful.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Colony PCR’ed selected Topo clones from Dpn1 treated and not treated.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran Colony PCR on a gel. Everything looks fine expect for Gal10/1* (Dpn1 treated) and Adh1p* (not Dpn1 treated). They don’t any PCR product.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>-Selected clones for minipreps, submitted them for sequencing using M13 REV primer.

<ul style='margin-top:0in' type=disc> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt;mso-list: l1 level1 lfo4;tab-stops:list .5in'>Selected clones Name (restriction    ends) Clone’s label:</li> <ul style='margin-top:0in' type=circle> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level2 lfo4;tab-stops:list 1.0in'>Dpn1 Treated</li> <ul style='margin-top:0in' type=square> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>TetR (x/b) A to C</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>TetR (AB) A to C</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>yEGFP (x/b) A to C</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>yEGFP (b/n) A to C</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>Adh1p* (p/x) A to C</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>Cyc1t (n/s) A to C</li> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>Gal10/1* (p/x) C</li> </ul> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level2 lfo4;tab-stops:list 1.0in'>Not treated with Dpn1</li> <ul style='margin-top:0in' type=square> <li class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt; mso-list:l1 level3 lfo4;tab-stops:list 1.5in'>Gal10/1* (p/x) A &amp; B</li> </ul> </ul> </ul>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'> 

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 3 (7/21/08 &#8211; 7/27/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>-Verified the Topo clones’ sequence and identified the good and bad clones.

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:.5in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list .5in'>-<span style='font-size:7.0pt;font-family:"Times New Roman"'> Good clones (all clones)

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      TetR (x/b)

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      Gal10/1* (p/x)

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      Cyc1t (n/s)

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:.5in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list .5in'>-<span style='font-size:7.0pt;font-family:"Times New Roman"'> Bad clones

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      TetR (AB) ALL clones.

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      yEGFP (x/b) ALL clones.

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      yEGFP (b/n) ALL clones.

<p class=MsoNormal style='margin-top:0in;margin-right:0in;margin-bottom:0in; margin-left:1.0in;margin-bottom:.0001pt;text-indent:-.25in;tab-stops:list 1.0in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      Adh1p* (p/x) ALL clones.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>-Repeated PCR on the bad clones expect TetR (AB) with exact extension time to prevent mutation. Dpn1 treated reaction

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Repeated the same PCR reaction as before.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran the PCR reaction on gel. There were no bands on the gel. Expect for a Adh1p*, but faint.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Repeated the same PCR reaction again. I diluted the template 20 fold.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran 2ul of the PCR reaction and the template on a gel. Everything looks ok.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Dpn1 treated the PCR reaction

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Selected clones from yEGFP (x/b), yEGFP (b/n), and Adh1p* (p/x) for miniprep and submitted them for sequencing using M13 REV primer.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran the PCR reactions that were Topo cloned on a gel to see if the Dpn1 treatment did anything to the PCR fragment. The fragments looked fine expect there are unknown bands.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Resent TetR (AB) clones for sequencing.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Verified TetR (AB) sequence. Every clone has 1 mutation.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- PCR’ed TetR (AB).

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Verified sequences for yEGFp (x/b), yEGFP (b/n), and Adh1p* (p/x). There are no good clones. However, I found that the mutations are all in the same spot. So I decided to proceed with Adh1p* (p/x) clone B and yEGFP (b/n) clone A.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Digest PAH 32 and Topo TetR (x/b) clone A with XhoI &amp; BamhI, PAH32 and Topo yEGFP (b/n) with BamhI &amp; NotI, PAH32 with XhoI &amp; NotI, AH81 AlexA, Topo Gal10/1* (p/x) clone A, and Topo Adh1p* (p/x) with PspomI &amp; XhoI.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel and gel purified the vector and insert above.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 4 (7/28/08 &#8211; 8/3/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>-Repeated digest Topo Adh1p* (p/x) with PspomI and XhoI again because yield was low, so I believe that I got the wrong gel slice.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel for Adh1p* (p/x) and gel purified.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Repeated PCR for for yEGFp (x/b), yEGFP (b/n), and Adh1p* (p/x) to prove my theory that the template contains the mutation and that it wasn’t caused by the PCR reaction.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel for TetR (x/b), yEGFp (x/b), yEGFP (b/n), and Adh1p* (p/x). Everything looks fine expect Adh1p* (p/x) fragment isn’t the right size. So I ran the gel longer and found that there are two bands on each lane. Jimmy said that someone moved the gel, so that could be the cause of the double bands. Also the ladder looks kind of squash together.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ligated TetR (x/b) into PAH32 vector, yEGFP (b/n) into PAH32 vector, Gal10/1*P (p/x) into AH81 AlexA vector, Adh1p* (p/x) into AH81 AlexA vector, and TetR (x/b) and yEGFP (b/n) into PAH32 vector.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Topo cloned TetR (AB), yEGFP (b/n), and yEGFP (x/b).

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Selected clones from TetR (x/b) + PAH32, yEGFP (b/n) + PAH32, Gal10/1*P (p/x) + AH81 AlexA, Ahd1p* (p/x) + AH81 AlexA, and TetR (x/b) and yEGFP (b/n) + PAH32 for miniprep and then test digest.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel for the test digest. Everything looks fine. Even the 3 part ligation looks correct.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Repeated test digest on TetR (x/b) and yEGFP (b/n) + PAH32. I just wanted to make sure it ligated correctly.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel for the test digest. Everything looks correct.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Selected clones from Topo clone TetR (AB), yEGFP (b/n), and yEGFP (x/b) for miniprep.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Digest PJAC9 (TetR (x/b) and yEGFP (b/n) + PAH32) with Xcm1.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Grow overnight culture, SV992 yeast strain for integration.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Digest PJAC9 with Xcm1 and more DNA and PJAC10 (Ahd1p* (p/x) + AH81 AlexA) and PJAC11 (Gal10/1*P (p/x) + AH81 AlexA) with Nhe1.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Transform PJAC9 PJAC10, PJAC11, PJAC9/PJAC10, and PJAC9/PJAC11 into SV992 yeast strain. PJAC9 is a PRS306 vector and PJAC10 and PJAC11 is a PRS303 vector. PRS306 vector are &#8211;URA and PRS303 is &#8211;HIS. For double transformation I plated them onto SD &#8211;HIS &#8211;URA plates.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'> 

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 5 (8/4/08 &#8211; 8/10/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Transformation efficiency was low so I place the cells in the incubator for an extra day.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Made ATC plates. Then restreak PJAC9/PJAC10 and PJAC9/PJAC11 onto the ATC plates.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Restreak PJAC9 onto SD &#8211;URA, PJAC10 and PJAC11 onto SD &#8211;HIS.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Setup culture for FACs on the Toggle switch stuff.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran FACs in block.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 6 (8/11/08 &#8211; 8/17/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Digest PJAC9, PAN023, and PAN0027 with PspomI &amp; XhoI.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel for the digest.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Repeated digest. (I believe that I forgot to add one enzyme)

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ran a gel for the digest and then gel purify.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Ligated Adh1p into PJAC 9 vector and Gal1 into PJAC9 vector.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Discuss the project with Lingli, so I can continue it.

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 7 (8/18/08 &#8211; 8/24/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>-

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<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>Week 8 (8/25/08 &#8211; 8/31/08)</b>

<p class=MsoNormal style='margin-bottom:0in;margin-bottom:.0001pt'>- Digest PJAC9, PJAC39 (Adh1p-TetR-GFP-Adh1T-LexOP), and PJAC40 (LexOP-Cyc1p-TetR-GFP-Adh1T) with NdeI and PJAC10 and PJAC11 with Xcm1

<p class=MsoNormal><![if !supportEmptyParas]> <![endif]></o:p>