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.

Milestones:

A)     Define any potential Protosilencers and insulators.

B)      Insert Protosilencer into different plasmids to see if it can help with distance silencing.

C)      Insert Insulator into different plasmids to see if it can stop silencing

Rational: Demonstrate the ability to control chromatin using Protosilencers and insulators.

Steps needed to accomplish objective.

1.      Find Protosilencers and insulator.

2.      PCR Protosilencer and Insulator

a.       Topo Clone.

b.      Sequence.

3.      PCR different length Spacers.

4.      Ligate Spacer into SAC1 site of PAH107.

a.       Quick Change an ASC1 site into the middle of the Spacers.

5.      Ligate either Protosilencer or Insulator into the KPN1 site of PAH107.

a.       Sequences insert to obtain both orientation of the Protosilencer/Insulator.

6.      Ligate either Protosilencer or Insulator into the ASC1 site of PAH107.

a.       Sequences insert to obtain both orientation of the Protosilencer/Insulator.

7.      Integrate complete plasmid into Gal1P-LexA-SIR2 (SV992) yeast strain.

8.      Test Protosilencer and Insulator on FACS.

 

Week 1 (7/28/08 – 8/3/08)

- PCRed protosilencer and insulator from genome DNA to see how large the parts are.

·        Protosilencer:

o       K19A; CoreX with Kpn1 ends.

o       A19A; CoreX with Asc1 ends.

o       K89A; Corex with Kpn1 ends.

o       A89A; CoreX with Asc1 ends.

·        Insulator

o       K19C; STR with Kpn1 ends.

o       A19A; STR with Asc1 ends.

- Repeated PCR, but with exact extension time.

- Ran a gel and found that two PCR reactions did not work.

- Repeated PCR with same extension time and another set with a longer extension time.

- Ran a gel. Same thing happen for the shorter extension time. However for the longer extension, it was able to PCR all the fragments.

- Repeated PCR with the shorter extension time for the last time.

- Ran a gel. Same thing happen, but one of failed PCR reaction from the previous reaction, worked.

- Topo Cloned.

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Week 2 (8/4/08 – 8/10/08)

- Selected clones for miniprep, and then submitted them for sequencing.

- Verified the Kpn1 ends Topo clones sequence and identify the best clone.

·        K19A clone 2

·        K89A clone 1

·        K19C clone 3

- 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.

- Repeated Digest for K19A clone 2.

-Ran a gel and I couldn’t see anything drop out.

- Ligated K89A1 into PAH107 vector and K19C3 into PAH107 vector.

- verified the ASC1 ends Topo clones sequence and identify the best clone.

·        A19A clone 2

·        A89A clone 1

·        A19C clone 2

- Selected clones from the ligation transformation for miniprep.

- PCR Y-Star with Kpn1 ends and Asc1 ends from genome DNA. Also PI3ka spacers with SacI ends from PI3ka plasmid.

- Test digested the ligation transformation clones. (K89A1+PAH107 and K19C3+PAH107.)

- Ran a gel. The reaction failed.

- Test digested again using Bsm1 to check for orientation of the insert.

- Ran a gel. The enzyme doesn’t seem to have cut the plasmid.

- Ran PCR reaction on a gel. PCR reaction for Y-STAR failed, but the PI3ka spacer worked expect that 1000bp spacer have two bands.

- Repeated PCR reaction for Y-STAR.

- Ran a gel

- Digest PAH107 with SacI.

- Gel purified K19A2 , S250, S500, S1000, and S2000.

- Digest S250, S500, S1000, and S2000 with Sac1.

- Ligated K19A2 into PAH107 vector.

- PCR purified S250, S500, S1000, and S2000.

- Gel purified PAH107.

- Ligated S250, S500, S1000, and S2000 into PAH107 vector.

- Selected colonies to miniprep and test digest.

- Test digested the ligation transformation with SacI for the spacers and Kpn1 for K19A2+PAH107.

-Ran Y-STAR Asc1 and Kpn1 PCR reaction.

- Topo cloned.

- Selected clones for miniprep.

- 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.

- Selected S250 clone 1, S500 clone 1, S1000 clone 1, and S2000 clone 1 to proceed.

-Quick change in an Asc1 site in between the spacers. Then transform into TG1 cells.

- Repeated ligation K19A2+PAH107.

 

Week 3 (8/11/08 – 8/17/08)

-Selected colonies from the ligation transformation for miniprep.

- Digested A19A2, A89A1, and A19C2 with Asc1.

- Repeated the Quick Changed transformation.

- Selected colonies for miniprep.

-Test digested PAH107 +K19A2, +K89A, and +K19C with Kpn1.

- 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.

- Sent PAH107 + K19A2 clones 2, 4, and 6 for sequencing.

- Repeat ligation for K89A1 into PAH107 vector using LIngli’s ligation system this time.

- Digest PAH107 + K19A2 with Kpn1 and relegated the together to get a different orientation.

- Test Digested Quick Changed Clones with ASC1.

- Ran a gel. I could see some linearization.

- Gel purified Spacers and Quick Changed Spacer vector.

- Ligated A19C, A19A, and A89A into Quick Changed Spacers vector.

- Verified sequence of PAH107+K19A2. Found out that all of the clones had the insert in the same orientation.

- Verified sequence of Y-STAR with Asc1 and Kpn1.

- Selected colonies for miniprep and submitted for sequencing using M13REV primer.

- Repeated Quick Change for Spacer 2000bp and transform.

- Selected colonies for miniprep.

- Test digested with Kpn1 and Quick Changed Spacer with Asc1.

- Ran a gel. PAH107 + K19A2 ligated, but PAH107+k89A didn’t worked. Also the Quick Changed Spacer 2000bp didn’t work.

- Digested PAH107+K19A with SacI.

- Repeated Quick Change for Spacer 2000bp, but this time I increase the extension time.

- Test digested with Kpn1 and BsmI. Also double digest with SacI and Asc1.

- Ran a gel. The Kpn1 and BsmI reaction didn’t work. However the double digest work, where I saw both spacer and insert.

- Digested QS(Quick Changed Spacer) 250.

- Ran a gel and couldn’t see the dropout fragment. .

 

Week 4 (8/18/08 – 8/24/08)

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Week 5 (8/25/08 – 8/31/08)

- Repeated test digest with Sac1.

- Ran a gel and everything looks fine.

- Digested Topo K89A and Topo K19C with Kpn1, and Qs250 with Sac1.

- Verified the orientation of PAH107+K19A and found two different orientation.

- 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.

- Selected more colonies from PAH107+QS500+A19A for miniprep and sent for sequencing using M13REV primer.

- Test Digested PAH107+K19A+QS(250,500, and 1000) (Forward/Reverse orientation) with SacI.

- Ran a gel. Everything looks fine.

- Test Digested PAH107+K19A+QS500 (F/R) with SacI.

- Digested plasmid with Xcm1.

- Transform digested plasmid into SV992 Gal1-LexA-Sir2 yeast strain.

- Verified sequences.

- Set colony PCR from the yeast transformation.

- Ran a gel for the colony PCR. Everything looked fine.

- Restreak colonies on Sraf -/+ Gal plates.

 

Week 6 (9/1/08 – 9/7/08)

- 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.

Week 7 (9/8/08 – 9/14/08)

- 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.

- FACS failed because I switch the media and cause weird data. Also the FACS stop working on the third block.

- 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.

- 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.

- 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.

Week 8 (9/15/08 – 9/21/08)

- Digested PAH107 + Spacer PAH107 + Spacer + A19C (F/R) and PAH32 with PspomI and Xho1.

- Gel purified.

- Ligated Cyc1P into PAH107 + Spacer PAH107 + Spacer + A19C (F/R) vector.

- Selected colonies for miniprep.

- Quick Changed an Asc1 site into PJH003 left and right.

- PCR’ed Fake insulator (STR and Y-STAR)

- Ran a gel for the Fake insulator.

- Test digested with Xho1 and PspomI.

 

Week 9 (9/22/08 – 9/28/08)

- Set up FACS to find the same level of expression of GFP for PAH107 + Spacers and PAH107 + Spacers + A19C (F/R).

- Ran FACS to find the same level of expression of GFP for PAH107 + Spacers and PAH107 + Spacers + A19C (F/R).

- Test digested with PspomI and xho1. Also Asc1 for Quick Changed PJH003 left and right plasmid.

- Ran 4 big and small gels.

- The DNA on the gels looked degraded.

- Ran a gel on the miniprep.

- Mini-preps are fine.

 

 

Week 10 (9/29/08 – 10/5/08)

- Digested with PsopmI and XhoI Cyc1p + PAH107 + Spacer + A19c (F/R)/ + NONE.

- Ran a gel.

- The Digest was successful. I can see the insert clearly and the DNA wasn’t degraded.

- Digested Cyc1p + PAH107 + Spacer + A19c (F/R)/ +NONE with XcmI for integration into yeast.

- Picked up colonies for Quick Changed PJH0003 1-1 for miniprep.

- Transformed Gal10-LexA-Sir2 (SV992) yeast strain. 

 

Week 11 (10/6/08 – 10/12/08)

- Digested Quick Change PJH0031-1 plasmid with AscI.

- Ran a gel. There were no sign of linearization.

- Repeated Digest and disgested Blank Y-STAR, Blank STR, and Topo A19c, and A42B.

- Ran a gel and then gel purified. There were no sign of linearization for Quick Changed PJH003 1-1.

- Mini-prep a different set of Quick Changed PJH003 1-1.

- Digested with AscI.

- Ran a Gel. The DNA looks degraded and there were no sign of linearization.

- Repeated digest.

- Ran a gel and there is one clone that linearized.

- Digest Quick ChangedPJH003 Left 5, Topo A19C, and A42B with AscI.

- Gel purified vector and insert.

- Digest AarI acceptor vector AD part with SacI and a different rxn with PspomI.

- Ligated Y-STAR/STR into Left 5 and then transformed.

- Picked colonies from the latest ligation transformation for mini-prep.

- Digest PJH003 1-1 with PspomI.

- CIP treated SACI digest of the AarI acceptor vector AD part.

- PCR’ed Y-STAR, STR, Blank Y-STAR, and Blank STR with PspomI ends.

- Ran a gel to see PCR’ed fragment.

- Topo cloned PCR’ed fragements.

- PCR purified PCR’ed fragments and then digested them with PspomI.

- Gel purifed AarI acceptor vector of SacI and PspomI rxn.

- Ligated the AarI acceptor vector with 8xLexA ops.

- Gel purifed PCR’ed fragement.

- Ligated PCR’ed fragments into PJH003 1-1.

 

Week 12 (10/13/08 – 10/19/08)

- Picked colonies form the latest ligation transformation for miniprep.

- Digested Y-STAR/STR/Blank STR+ Left5 with AscI.

- PCR’ed 3000bp Spacer from PI3Ka plasmid.

- Digested PCR’ed fragment+ PJH00031-1 with PspomI.

- Digested LexA + AarI acceptor vector AD part with PspomI and Xho1 and another rxn with NotI and then SacI.

- 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.

- Ran a gel for PCR’ed 3000bp Spacer. There were no bands.

- Repeated PCR rxn for 3000bp Spacer, but this time I going to do a temperature gradient.

- Ran a gel. The lowest annealing temperature had a 3000bp band.

- Repeated Digest for Y-STAR/STR/Blank STR+ Left5 and PCR’ed fragment+ PJH00031-1.

- Digested topo clones with PspomI and S2000 with SacI.

 

Week 13 (10/20/08 – 10/26/08)

- Verified the sequence of the insert 8xLexAops in the AarI acceptor vector AD parts.

- Ran a gel for last week digest. There were no insert dropouts.

- Digested PCR 3000bp spacer, S2000, and Cyc1p + PAH107+QS250 with SacI.

- Gel purified insert and vector.

- Ligated the 3000bp Spacer/S2000 into Cyc1p +PAH107 vector.

- Picked colonies for miniprep.

- Test digest with Saci.

- Digest 3000bp Spacer/S2000 + Cyc1P + PAH107 with XcmI for yeast integration.

- Transformed Gal10P-LexA-Sir2 (Sv992) yeast strain.

- Digested Fig1p AarI acceptor vector with PspomI and another rxn with SacI.

- PCR’ed GFP AD.

- Ran a gel and gel puified Fig1p AarI acceptor vector.

- Topo cloned GFP AD.

- Ligated SacI/PspomI LexAops into Fig1p AarI acceptor vector.

- Streak transformed yeast onto SD-Ura.

- Picked colonies for miniprep.

- Digested SacI/PspomI LexAops + Fig1p AarI acceptor vector with SacI and then NotI and another rxn with PspomI and XhoI.

- Ran gel. PspomI and XhoI rxn was successful, but SacI and NotI rxn was not. I believe that one enzyme didn’t work.

- Sent SacI/PspomI LexAops + Fig1p AarI acceptor vector and Topo GFP AD plasmid for sequence.

- Restreak transformed yeasted on Sraf +/- 2% Gal.

 

 

Week 14 (10/27/08 – 11/2/08)

- Verified the sequence of SacI/PspomI LexAops + Fig1p AarI acceptor vector.