Editing PLentiGP-Cre (section)

= Repeat  =

The following exp was done by Paul<br> 

== PCR  ==

[[Image:Cre PCR 2011 7 10.jpg]] 

Amplify Cre using Lenti-Cre-F + Lenti-Cre-R, template: pTriEx-HTNC, 25 cycles, TurboPfu 

Run gel and purify PCR fragment. 

--[[User:Paul.xu|Paul.xu]] 16:26, 10 July 2011 (EST) 

== Cloning<br>  ==

[[Image:PLentiGP pLVGP EcoR1 Xho1 Nde1 Cre 2011 7 10.jpg]]<br> <br> 1: pLVGP, 6-17-2011, EcoR1+Xho1+Nde1<br> 

2: pLentiGP, 6-16-2011, #2, EcoR1+Xho1+Nde1<br> 

3: pLentiGP, 6-16-2011, #1, EcoR1+Xho1+Nde1<br> 

4: pLentiPlus, #3, EcoR1+Xho1+Nde1<br> 

5: pLVGP, EcoR1+Xho1<br> 

6: pLentiPlus, EcoR1+Xho1<br> 

7: Cre PCR fragment, EcoR1+Xho1<br> 

8: Cre PCR fragment,, EcoR1+Xho1<br> 

Purify vectors #5 &amp; #6 

Purify PCR fragment, #7 &amp; #8 

Ligate#5 + #7, #6+#8. Transform STBL3, grow on LB/Amp o/n<br> 

--[[User:Paul.xu|Paul.xu]] 16:08, 10 July 2011 (EST) 

<br> 

[[Image:IMG 1083.JPG]]<br> 

Line 1: PlentiPlus clone ID: 219 (tube 1), NdeI + EcoR1<br>Line 2: PlentiPlus clone ID: 219 (tube 2), NdeI + EcoR1<br>Line 3: PlentiPlus clone ID: 219 (tube 3), NdeI + EcoR1<br>Line 4: PlentiPlus Amp 3 , NdeI + EcoR1<br>Conclusion: MCS is intack. 

<br> 

--[[User:Paul.xu|Paul.xu]] 16:08, 10 July 2011 (EST) 

<br> 

[[Image:242+300.JPG]]<br> <br> 1: Marker<br> 

2: pAd5-CMV-2-IRES-GFP (ID, 242),#1, Xba1+Nde1<br> 

3: pAd5-CMV-2-IRES-GFP(ID, 242),#2, Xba1+Nde1<br> 

4: pAd5-CMV-2-IRES-GFP(ID, 242),#3, Xba1+Nde11<br> 

5: pAd5-CMV-2-IRES-GFP(ID, 242),#4, Xba1+Nde1<br> 

6: pAd5-CMV-2-IRES-GFP(ID, 242),#5, Xba1+Nde1<br> 

7: pLenti-PKA(ID, 300), #1,Nhe1+Xho1<br> 

8: pLenti-PKA(ID, 300), #2,Nhe1+Xho1<br> 

Sequencing: pAd5-CMV-2-IRES-GFP(ID, 242),#2;<br> 

<br> 

[[Image:252+254.JPG]]<br> <br> 1: Marker<br> 2: plenti-pka(ID, 300), #3,Nhe1+Xho1<br> 

3: plenti-pka(ID, 300), #4,Nhe1+Xho1<br> 

4: pAAV-MCS(ID, 254) ,#1, KpnI+Nco1<br> 

5: pAAV-MCS(ID, 254) ,#2, KpnI+Nco1<br> 

6: pAAV-MCS(ID, 254) ,#3, KpnI+Nco1<br> 

7: pAAV-MCS(ID, 254) ,#4, KpnI+Nco1<br> 

8: pAAV-MCS(ID, 254) ,#5, KpnI+Nco1<br> 

Sequencing: pLenti-pka(ID, 300), #3<br> pAAV-MCS(ID, 254) ,#2<br> 

<br> [[Image:254+280.JPG]]<br> <br> 1: Marker<br> 2: pAAV-MCS(ID, 254) ,#1, KpnI+Nco1<br> 

3: pAAV-MCS(ID, 254) ,#2, KpnI+Nco1<br> 

4: pAAV-MCS(ID, 254) ,#3, KpnI+Nco1<br> 

5: pAAV-MCS(ID, 254) ,#4, KpnI+Nco1<br> 

6: pAd5-CMV-MCS(ID, 280) ,#1, NdeI+XbaI<br> 

7: pAd5-CMV-MCS(ID, 280) ,#2, NdeI+XbaI<br> 

8: pAd5-CMV-MCS(ID, 280) ,#3, NdeI+XbaI<br> 9: Marker<br> 10: pAd5-CMV-MCS(ID, 280) ,#4, NdeI+XbaI<br> 11: pAd5-CMV-MCS(ID, 280) ,#5, NdeI+XbaI<br> 

sequencing: pAd5-CMV-MCS(ID, 280) ,#1<br> 

2011-07-21 Haiyan, for pet his-cre and 256 colonies creening<br>
<br> [[Image:CRE-0721.JPG]]<br> <br> 1: Marker<br> 2: PET 8His(ID, 286) , Xhoi+Nco1<br> 

3: plentiCre(ID, 241) ,Xhoi+Nco1<br> 

4: pAAV-cre(ID, 256) ,#1, ecori+xhoi<br> 

5: pAAV-cre(ID, 256) ,#2, ecori+xhoi<br> 

6: pAAV-cre(ID, 256) ,#3, ecori+xhoi<br> 

ligate 2 &3; colonies screening for 256, 5 and 6 should be positive.


<br> Digestion and gel 


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[[Category:Cloning]]