Side Project 2--Insulators, Boundary Elements and Spreading



       Objective 2: Determine if Protosilencer can help increase the distance of silencing and if insulator can interrupt silencing

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 Objective 2: Determine if Protosilencer can help increase the distance of silencing and if insulator can interrupt silencing. 

Milestones:

A)<span style='font-size: 7.0pt;font-family:"Times New Roman"'>    Define any potential Protosilencers and insulators.

<p class=listparagraph style='text-indent:-.25in'>B)<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Insert Protosilencer into different plasmids to see if it can help with distance silencing.

<p class=listparagraph style='text-indent:-.25in'>C)<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Insert Insulator into different plasmids to see if it can stop silencing

<p class=MsoNormal>Rational: Demonstrate the ability to control chromatin using Protosilencers and insulators.

<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"'>     Find Protosilencers and insulator.

<p class=listparagraph style='text-indent:-.25in'>2.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     PCR Protosilencer and Insulator

<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.

<p class=listparagraph style='text-indent:-.25in'>3.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     PCR different length Spacers.

<p class=listparagraph style='text-indent:-.25in'>4.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Ligate Spacer into SAC1 site of PAH107.

<p class=listparagraph style='margin-left:1.0in;text-indent:-.25in'>a.<span style='font-size:7.0pt;font-family:"Times New Roman"'> Quick Change an ASC1 site into the middle of the Spacers.

<p class=listparagraph style='text-indent:-.25in'>5.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Ligate either Protosilencer or Insulator into the KPN1 site of PAH107.

<p class=listparagraph style='margin-left:1.0in;text-indent:-.25in'>a.<span style='font-size:7.0pt;font-family:"Times New Roman"'> Sequences insert to obtain both orientation of the Protosilencer/Insulator.

<p class=listparagraph style='text-indent:-.25in'>6.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Ligate either Protosilencer or Insulator into the ASC1 site of PAH107.

<p class=listparagraph style='margin-left:1.0in;text-indent:-.25in'>a.<span style='font-size:7.0pt;font-family:"Times New Roman"'> Sequences insert to obtain both orientation of the Protosilencer/Insulator.

<p class=listparagraph style='text-indent:-.25in'>7.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Integrate complete plasmid into Gal1P-LexA-SIR2 (SV992) yeast strain.

<p class=listparagraph style='text-indent:-.25in'>8.<span style='font-size: 7.0pt;font-family:"Times New Roman"'>     Test Protosilencer and Insulator on FACS.

<p class=listparagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=listparagraph style='margin-left:0in'>Week 1 (7/28/08 &#8211; 8/3/08)</b>

<p class=listparagraph style='margin-left:0in'>- PCRed protosilencer and insulator from genome DNA to see how large the parts are.

<p class=listparagraph style='margin-left:37.45pt;text-indent:-.25in'><span style='font-family:Symbol'>· <span style='font-size:7.0pt;font-family: "Times New Roman"'>       Protosilencer:

<p class=listparagraph style='margin-left:73.45pt;text-indent:-.25in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      K19A; CoreX with Kpn1 ends.

<p class=listparagraph style='margin-left:73.45pt;text-indent:-.25in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      A19A; CoreX with Asc1 ends.

<p class=listparagraph style='margin-left:73.45pt;text-indent:-.25in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      K89A; Corex with Kpn1 ends.

<p class=listparagraph style='margin-left:73.45pt;text-indent:-.25in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      A89A; CoreX with Asc1 ends.

<p class=listparagraph style='margin-left:37.45pt;text-indent:-.25in'><span style='font-family:Symbol'>· <span style='font-size:7.0pt;font-family: "Times New Roman"'>       Insulator

<p class=listparagraph style='margin-left:73.45pt;text-indent:-.25in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      K19C; STR with Kpn1 ends.

<p class=listparagraph style='margin-left:73.45pt;text-indent:-.25in'><span style='font-family:"Courier New"'>o <span style='font-size:7.0pt; font-family:"Times New Roman"'>      A19A; STR with Asc1 ends.

<p class=listparagraph style='margin-left:0in'>- Repeated PCR, but with exact extension time.

<p class=listparagraph style='margin-left:0in'>- Ran a gel and found that two PCR reactions did not work.

<p class=listparagraph style='margin-left:0in'>- Repeated PCR with same extension time and another set with a longer extension time.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. Same thing happen for the shorter extension time. However for the longer extension, it was able to PCR all the fragments.

<p class=listparagraph style='margin-left:0in'>- Repeated PCR with the shorter extension time for the last time.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. Same thing happen, but one of failed PCR reaction from the previous reaction, worked.

<p class=listparagraph style='margin-left:0in'>- Topo Cloned.

<p class=listparagraph style='margin-left:0in'>-

<p class=listparagraph style='margin-left:0in'>Week 2 (8/4/08 &#8211; 8/10/08)</b>

<p class=listparagraph style='margin-left:0in'>- Selected clones for miniprep, and then submitted them for sequencing.

<p class=listparagraph style='margin-left:0in'>- Verified the Kpn1 ends Topo clones sequence and identify the best clone.

<p class=listparagraph style='text-indent:-.25in'><span style='font-family: Symbol'>· K19A clone 2

<p class=listparagraph style='text-indent:-.25in'><span style='font-family: Symbol'>· K89A clone 1

<p class=listparagraph style='text-indent:-.25in'><span style='font-family: Symbol'>· K19C clone 3

<p class=listparagraph style='margin-left:0in'>- Digest Kpn1 ends Topo clones and PAH107 with Kpn1. - Ran a gel and then gel purify. Expect for K19A2 because I can’t really see the band.

<p class=listparagraph style='margin-left:0in'>- Repeated Digest for K19A clone 2.

<p class=listparagraph style='margin-left:0in'>-Ran a gel and I couldn’t see anything drop out.

<p class=listparagraph style='margin-left:0in'>- Ligated K89A1 into PAH107 vector and K19C3 into PAH107 vector.

<p class=listparagraph style='margin-left:0in'>- verified the ASC1 ends Topo clones sequence and identify the best clone.

<p class=listparagraph style='text-indent:-.25in'><span style='font-family: Symbol'>· A19A clone 2

<p class=listparagraph style='text-indent:-.25in'><span style='font-family: Symbol'>· A89A clone 1

<p class=listparagraph style='text-indent:-.25in'><span style='font-family: Symbol'>· A19C clone 2

<p class=listparagraph style='margin-left:0in'>- Selected clones from the ligation transformation for miniprep.

<p class=listparagraph style='margin-left:0in'>- PCR Y-Star with Kpn1 ends and Asc1 ends from genome DNA. Also PI3ka spacers with SacI ends from PI3ka plasmid.

<p class=listparagraph style='margin-left:0in'>- Test digested the ligation transformation clones. (K89A1+PAH107 and K19C3+PAH107.)

<p class=listparagraph style='margin-left:0in'>- Ran a gel. The reaction failed.

<p class=listparagraph style='margin-left:0in'>- Test digested again using Bsm1 to check for orientation of the insert.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. The enzyme doesn’t seem to have cut the plasmid.

<p class=listparagraph style='margin-left:0in'>- Ran PCR reaction on a gel. PCR reaction for Y-STAR failed, but the PI3ka spacer worked expect that 1000bp spacer have two bands.

<p class=listparagraph style='margin-left:0in'>- Repeated PCR reaction for Y-STAR.

<p class=listparagraph style='margin-left:0in'>- Ran a gel

<p class=listparagraph style='margin-left:0in'>- Digest PAH107 with SacI.

<p class=listparagraph style='margin-left:0in'>- Gel purified K19A2, S250, S500, S1000, and S2000.

<p class=listparagraph style='margin-left:0in'>- Digest S250, S500, S1000, and S2000 with Sac1.

<p class=listparagraph style='margin-left:0in'>- Ligated K19A2 into PAH107 vector.

<p class=listparagraph style='margin-left:0in'>- PCR purified S250, S500, S1000, and S2000.

<p class=listparagraph style='margin-left:0in'>- Gel purified PAH107.

<p class=listparagraph style='margin-left:0in'>- Ligated S250, S500, S1000, and S2000 into PAH107 vector.

<p class=listparagraph style='margin-left:0in'>- Selected colonies to miniprep and test digest.

<p class=listparagraph style='margin-left:0in'>- Test digested the ligation transformation with SacI for the spacers and Kpn1 for K19A2+PAH107.

<p class=listparagraph style='margin-left:0in'>-Ran Y-STAR Asc1 and Kpn1 PCR reaction.

<p class=listparagraph style='margin-left:0in'>- Topo cloned.

<p class=listparagraph style='margin-left:0in'>- Selected clones for miniprep.

<p class=listparagraph style='margin-left:0in'>- Ran a gel for the test digest. For the K19A2+PAH107 digest with Kpn1. There are no bands, not even plasmid. I believe that I miniprep containment. For the spacer digest, everything looks fine. There are 2 clones that doesn’t the insert.

<p class=listparagraph style='margin-left:0in'>- Selected S250 clone 1, S500 clone 1, S1000 clone 1, and S2000 clone 1 to proceed.

<p class=listparagraph style='margin-left:0in'>-Quick change in an Asc1 site in between the spacers. Then transform into TG1 cells.

<p class=listparagraph style='margin-left:0in'>- Repeated ligation K19A2+PAH107.

<p class=listparagraph style='margin-left:0in'>

<p class=listparagraph style='margin-left:0in'>Week 3 (8/11/08 &#8211; 8/17/08)</b>

<p class=listparagraph style='margin-left:0in'>-Selected colonies from the ligation transformation for miniprep.

<p class=listparagraph style='margin-left:0in'>- Digested A19A2, A89A1, and A19C2 with Asc1.

<p class=listparagraph style='margin-left:0in'>- Repeated the Quick Changed transformation.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep.

<p class=listparagraph style='margin-left:0in'>-Test digested PAH107 +K19A2, +K89A, and +K19C with Kpn1.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. For PAH107 + K19A2, everything is fine. For PAH107 + K89A, there was a band drop out so failed. For PAH107 + K19C, there seems to be two bands.

<p class=listparagraph style='margin-left:0in'>- Sent PAH107 + K19A2 clones 2, 4, and 6 for sequencing.

<p class=listparagraph style='margin-left:0in'>- Repeat ligation for K89A1 into PAH107 vector using LIngli’s ligation system this time.

<p class=listparagraph style='margin-left:0in'>- Digest PAH107 + K19A2 with Kpn1 and relegated the together to get a different orientation.

<p class=listparagraph style='margin-left:0in'>- Test Digested Quick Changed Clones with ASC1.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. I could see some linearization.

<p class=listparagraph style='margin-left:0in'>- Gel purified Spacers and Quick Changed Spacer vector.

<p class=listparagraph style='margin-left:0in'>- Ligated A19C, A19A, and A89A into Quick Changed Spacers vector.

<p class=listparagraph style='margin-left:0in'>- Verified sequence of PAH107+K19A2. Found out that all of the clones had the insert in the same orientation.

<p class=listparagraph style='margin-left:0in'>- Verified sequence of Y-STAR with Asc1 and Kpn1.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep and submitted for sequencing using M13REV primer.

<p class=listparagraph style='margin-left:0in'>- Repeated Quick Change for Spacer 2000bp and transform.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep.

<p class=listparagraph style='margin-left:0in'>- Test digested with Kpn1 and Quick Changed Spacer with Asc1.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. PAH107 + K19A2 ligated, but PAH107+k89A didn’t worked. Also the Quick Changed Spacer 2000bp didn’t work.

<p class=listparagraph style='margin-left:0in'>- Digested PAH107+K19A with SacI.

<p class=listparagraph style='margin-left:0in'>- Repeated Quick Change for Spacer 2000bp, but this time I increase the extension time.

<p class=listparagraph style='margin-left:0in'>- Test digested with Kpn1 and BsmI. Also double digest with SacI and Asc1.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. The Kpn1 and BsmI reaction didn’t work. However the double digest work, where I saw both spacer and insert.

<p class=listparagraph style='margin-left:0in'>- Digested QS(Quick Changed Spacer) 250.

<p class=listparagraph style='margin-left:0in'>- Ran a gel and couldn’t see the dropout fragment. .

<p class=listparagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=listparagraph style='margin-left:0in'>Week 4 (8/18/08 &#8211; 8/24/08)</b>

<p class=listparagraph style='margin-left:0in'>-

<p class=listparagraph style='margin-left:0in'>Week 5 (8/25/08 &#8211; 8/31/08)</b>

<p class=listparagraph style='margin-left:0in'>- Repeated test digest with Sac1.

<p class=listparagraph style='margin-left:0in'>- Ran a gel and everything looks fine.

<p class=listparagraph style='margin-left:0in'>- Digested Topo K89A and Topo K19C with Kpn1, and Qs250 with Sac1.

<p class=listparagraph style='margin-left:0in'>- Verified the orientation of PAH107+K19A and found two different orientation.

<p class=listparagraph style='margin-left:0in'>- Verified the orientation of PAH107+QS250+A19A/A89A/A19C, PAH107+QS500+A19A/A89A/A19C, PAH107+QS1000+A19A/A89A/A19C. Every set of ligation had two orientations expect for PAH107+QS500+A19A.

<p class=listparagraph style='margin-left:0in'>- Selected more colonies from PAH107+QS500+A19A for miniprep and sent for sequencing using M13REV primer.

<p class=listparagraph style='margin-left:0in'>- Test Digested PAH107+K19A+QS(250,500, and 1000) (Forward/Reverse orientation) with SacI.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. Everything looks fine.

<p class=listparagraph style='margin-left:0in'>- Test Digested PAH107+K19A+QS500 (F/R) with SacI.

<p class=listparagraph style='margin-left:0in'>- Digested plasmid with Xcm1.

<p class=listparagraph style='margin-left:0in'>- Transform digested plasmid into SV992 Gal1-LexA-Sir2 yeast strain.

<p class=listparagraph style='margin-left:0in'>- Verified sequences.

<p class=listparagraph style='margin-left:0in'>- Set colony PCR from the yeast transformation.

<p class=listparagraph style='margin-left:0in'>- Ran a gel for the colony PCR. Everything looked fine.

<p class=listparagraph style='margin-left:0in'>- Restreak colonies on Sraf -/+ Gal plates.

<p class=listparagraph style='margin-left:0in'> 

<p class=listparagraph style='margin-left:0in'>Week 6 (9/1/08 &#8211; 9/7/08)</b>

<p class=listparagraph style='margin-left:0in'>- Set up FACS for testing the affect of the Insulator and Protosilencer on silencing. Also if the distance or orientation would cause any change in the effect.

<p class=listparagraph style='margin-left:0in'>Week 7 (9/8/08 &#8211; 9/14/08)</b>

<p class=listparagraph style='margin-left:0in'>- Ran FACS for testing the affect of the Insulator and Protosilencer on silencing. Also if the distance or orientation would cause any change in the effect.

<p class=listparagraph style='margin-left:0in'>- FACS failed because I switch the media and cause weird data. Also the FACS stop working on the third block.

<p class=listparagraph style='margin-left:0in'>- Repeated FACS again. Set up culture for testing the affect of the Insulator and Protosilencer on silencing. Also if the distance or orientation would cause any change in the effect.

<p class=listparagraph style='margin-left:0in'>- Ran FACS for testing the affect of the Insulator and Protosilencer on silencing. Also if the distance or orientation would cause any change in the effect.

<p class=listparagraph style='margin-left:0in'>- From the FACS data, I concluded that the protosilencer doesn’t work or that it need to interact with the really silencers. So this project will proceed with insulator.

<p class=listparagraph style='margin-left:0in'>Week 8 (9/15/08 &#8211; 9/21/08)</o:p></b>

<p class=listparagraph style='margin-left:0in'>- Digested PAH107 + Spacer PAH107 + Spacer + A19C (F/R) and PAH32 with PspomI and Xho1.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Ligated Cyc1P into PAH107 + Spacer PAH107 + Spacer + A19C (F/R) vector.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep.

<p class=listparagraph style='margin-left:0in'>- Quick Changed an Asc1 site into PJH003 left and right.

<p class=listparagraph style='margin-left:0in'>- PCR’ed Fake insulator (STR and Y-STAR)

<p class=listparagraph style='margin-left:0in'>- Ran a gel for the Fake insulator.

<p class=listparagraph style='margin-left:0in'>- Test digested with Xho1 and PspomI.

<p class=listparagraph style='margin-left:0in'>

<p class=listparagraph style='margin-left:0in'>Week 9 (9/22/08 &#8211; 9/28/08)</o:p></b>

<p class=listparagraph style='margin-left:0in'>- Set up FACS to find the same level of expression of GFP for PAH107 + Spacers and PAH107 + Spacers + A19C (F/R).

<p class=listparagraph style='margin-left:0in'>- Ran FACS to find the same level of expression of GFP for PAH107 + Spacers and PAH107 + Spacers + A19C (F/R).

<p class=listparagraph style='margin-left:0in'>- Test digested with PspomI and xho1. Also Asc1 for Quick Changed PJH003 left and right plasmid.

<p class=listparagraph style='margin-left:0in'>- Ran 4 big and small gels.

<p class=ListParagraph style='margin-left:0in'>- The DNA on the gels looked degraded.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel on the miniprep.

<p class=ListParagraph style='margin-left:0in'>- Mini-preps are fine.

<p class=ListParagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=ListParagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]></o:p>

<p class=ListParagraph style='margin-left:0in'>Week 10 (9/29/08 &#8211; 10/5/08)<o:p></o:p></b>

<p class=ListParagraph style='margin-left:0in'>- Digested with PsopmI and XhoI Cyc1p + PAH107 + Spacer + A19c (F/R)/ + NONE.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel.

<p class=ListParagraph style='margin-left:0in'>- The Digest was successful. I can see the insert clearly and the DNA wasn’t degraded.

<p class=ListParagraph style='margin-left:0in'>- Digested Cyc1p + PAH107 + Spacer + A19c (F/R)/ +NONE with XcmI for integration into yeast.

<p class=ListParagraph style='margin-left:0in'>- Picked up colonies for Quick Changed PJH0003 1-1 for miniprep.

<p class=ListParagraph style='margin-left:0in'>- Transformed Gal10-LexA-Sir2 (SV992) yeast strain.

<p class=ListParagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=ListParagraph style='margin-left:0in'><b>Week 11 (10/6/08 &#8211; 10/12/08)<o:p></o:p></b>

<p class=ListParagraph style='margin-left:0in'>- Digested Quick Change PJH0031-1 plasmid with AscI.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel. There were no sign of linearization.

<p class=ListParagraph style='margin-left:0in'>- Repeated Digest and disgested Blank Y-STAR, Blank STR, and Topo A19c, and A42B.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel and then gel purified. There were no sign of linearization for Quick Changed PJH003 1-1.

<p class=ListParagraph style='margin-left:0in'>- Mini-prep a different set of Quick Changed PJH003 1-1.

<p class=ListParagraph style='margin-left:0in'>- Digested with AscI.

<p class=ListParagraph style='margin-left:0in'>- Ran a Gel. The DNA looks degraded and there were no sign of linearization.

<p class=ListParagraph style='margin-left:0in'>- Repeated digest.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel and there is one clone that linearized.

<p class=ListParagraph style='margin-left:0in'>- Digest Quick ChangedPJH003 Left 5, Topo A19C, and A42B with AscI.

<p class=ListParagraph style='margin-left:0in'>- Gel purified vector and insert.

<p class=ListParagraph style='margin-left:0in'>- Digest AarI acceptor vector AD part with SacI and a different rxn with PspomI.

<p class=ListParagraph style='margin-left:0in'>- Ligated Y-STAR/STR into Left 5 and then transformed.

<p class=ListParagraph style='margin-left:0in'>- Picked colonies from the latest ligation transformation for mini-prep.

<p class=ListParagraph style='margin-left:0in'>- Digest PJH003 1-1 with PspomI.

<p class=ListParagraph style='margin-left:0in'>- CIP treated SACI digest of the AarI acceptor vector AD part.

<p class=ListParagraph style='margin-left:0in'>- PCR’ed Y-STAR, STR, Blank Y-STAR, and Blank STR with PspomI ends.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel to see PCR’ed fragment.

<p class=ListParagraph style='margin-left:0in'>- Topo cloned PCR’ed fragements.

<p class=ListParagraph style='margin-left:0in'>- PCR purified PCR’ed fragments and then digested them with PspomI.

<p class=ListParagraph style='margin-left:0in'>- Gel purifed AarI acceptor vector of SacI and PspomI rxn.

<p class=ListParagraph style='margin-left:0in'>- Ligated the AarI acceptor vector with 8xLexA ops.

<p class=ListParagraph style='margin-left:0in'>- Gel purifed PCR’ed fragement.

<p class=ListParagraph style='margin-left:0in'>- Ligated PCR’ed fragments into PJH003 1-1.

<p class=ListParagraph style='margin-left:0in'><span style="mso-spacerun: yes">

<p class=ListParagraph style='margin-left:0in'><b>Week 12 (10/13/08 &#8211; 10/19/08)<o:p></o:p></b>

<p class=ListParagraph style='margin-left:0in'>- Picked colonies form the latest ligation transformation for miniprep.

<p class=ListParagraph style='margin-left:0in'>- Digested Y-STAR/STR/Blank STR+ Left5 with AscI.

<p class=ListParagraph style='margin-left:0in'>- PCR’ed 3000bp Spacer from PI3Ka plasmid.

<p class=ListParagraph style='margin-left:0in'>- Digested PCR’ed fragment+ PJH00031-1 with PspomI.

<p class=ListParagraph style='margin-left:0in'>- Digested LexA + AarI acceptor vector AD part with PspomI and Xho1 and another rxn with NotI and then SacI.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel. The bands are very weak and faint for the Y-STAR/STR/Blank STR+ Left5 and PCR’ed fragment+ PJH00031-1 digest. For the PspomI and XhoI and NotI and SacI digests looks fine.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel for PCR’ed 3000bp Spacer. There were no bands.

<p class=ListParagraph style='margin-left:0in'>- Repeated PCR rxn for 3000bp Spacer, but this time I going to do a temperature gradient.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel. The lowest annealing temperature had a 3000bp band.

<p class=ListParagraph style='margin-left:0in'>- Repeated Digest for Y-STAR/STR/Blank STR+ Left5 and PCR’ed fragment+ PJH00031-1.

<p class=ListParagraph style='margin-left:0in'>- Digested topo clones with PspomI and S2000 with SacI.

<p class=ListParagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=ListParagraph style='margin-left:0in'><b>Week 13 (10/20/08 &#8211; 10/26/08)<o:p></o:p></b>

<p class=ListParagraph style='margin-left:0in'>- Verified the sequence of the insert 8xLexAops in the AarI acceptor vector AD parts.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel for last week digest. There were no insert dropouts.

<p class=ListParagraph style='margin-left:0in'>- Digested PCR 3000bp spacer, S2000, and Cyc1p + PAH107+QS250 with SacI.

<p class=ListParagraph style='margin-left:0in'>- Gel purified insert and vector.

<p class=ListParagraph style='margin-left:0in'>- Ligated the 3000bp Spacer/S2000 into Cyc1p +PAH107 vector.

<p class=ListParagraph style='margin-left:0in'>- Picked colonies for miniprep.

<p class=ListParagraph style='margin-left:0in'>- Test digest with Saci.

<p class=ListParagraph style='margin-left:0in'>- Digest 3000bp Spacer/S2000 + Cyc1P + PAH107 with XcmI for yeast integration.

<p class=ListParagraph style='margin-left:0in'>- Transformed Gal10P-LexA-Sir2 (Sv992) yeast strain.

<p class=ListParagraph style='margin-left:0in'>- Digested Fig1p AarI acceptor vector with PspomI and another rxn with SacI.

<p class=ListParagraph style='margin-left:0in'>- PCR’ed GFP AD.

<p class=ListParagraph style='margin-left:0in'>- Ran a gel and gel puified Fig1p AarI acceptor vector.

<p class=ListParagraph style='margin-left:0in'>- Topo cloned GFP AD.

<p class=ListParagraph style='margin-left:0in'>- Ligated SacI/PspomI LexAops into Fig1p AarI acceptor vector.

<p class=ListParagraph style='margin-left:0in'>- Streak transformed yeast onto SD-Ura.

<p class=ListParagraph style='margin-left:0in'>- Picked colonies for miniprep.

<p class=ListParagraph style='margin-left:0in'>- Digested SacI/PspomI LexAops + Fig1p AarI acceptor vector with SacI and then NotI and another rxn with PspomI and XhoI.

<p class=ListParagraph style='margin-left:0in'>- Ran gel. PspomI and XhoI rxn was successful, but SacI and NotI rxn was not. I believe that one enzyme didn’t work.

<p class=ListParagraph style='margin-left:0in'>- Sent SacI/PspomI LexAops + Fig1p AarI acceptor vector and Topo GFP AD plasmid for sequence.

<p class=ListParagraph style='margin-left:0in'>- Restreak transformed yeasted on Sraf +/- 2% Gal.

<p class=ListParagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=ListParagraph style='margin-left:0in'><![if !supportEmptyParas]> <![endif]><o:p></o:p>

<p class=ListParagraph style='margin-left:0in'><b>Week 14 (10/27/08 &#8211; 11/2/08)<o:p></o:p></b>

<p class=ListParagraph style='margin-left:0in'>- Verified the sequence of SacI/PspomI LexAops + Fig1p AarI acceptor vector.

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