Hylei at 17:59, 21 July 2011

2011-07-21T17:59:41Z

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= '''Cloning of Cre to pLentiGP''' =

Purpose: cloning of Cre to pLentiGP 

Date: 2011-6-28 to 2011-6-29 (2011-7-5 to 2011-7-8)

Notebook:Ruili Su (haiyan lei)

Page:002788--002789

Clone ID:# 241 

Experimenters:  Ruili 

== '''PCR''' ==

Date: 2011-6-28

PCR to amplify Cre

[[Image:PlentiGP--LoxP-06-28-11-1.JPG]]

1. Marker

3. Amplified Cre fragment from PCR.

== '''Cloning''' ==

Digest PlentiGP (clone 219) with EcoRI+ XhoI 

Digest LoxP fregment with EcoRI+ XhoI 

cut out insert and vector, ligate and transform STBL3 

 

[[Image:PlentiGP--LoxP-06-28-11-2.JPG]] 

1. Marker. 

4. Digested PlentiGP (clone 219) with ECoRI+ XHoI 

5. Digested Cre fragment with ECoRI+ XHoI 

[[Image:PlentiGP--LoxP-06-28-11-2.JPG]] 

Line 2: PlentiGP/ECoRI+ XHoI (241 Cre vector) Line 3: PlentiGP/BamHI+ XHoI (259 Loxp vector) Line 4: PlentiGP/NheI+ XHoI (pka vector) Line 5: pAAV-GFP/EcoRI+ XHoI (254 pAAV-MCS vector) Line 6: 10ul Cre PCR amplified product (50ul volume) Line 7: 10ul Loxp PCR amplified product (50ul volume) 

[[Image:Pka-070811.JPG]] 

 

hylei, 07-08-11

== Result intepretation/Summary ==

The line 4 and line 5 show digestion with ECoRI+ XHoI

The line 5 (digested Cre fragment with ECoRI+ XHoI) shows the same size as the line 3 (amplified Cre fragment from PCR).

== Conclusion ==

The digestion is complete and good, [[User:Ruili.su|Ruili.su]] 14:52, 28 June 2011 (EST)

== Comments ==

clone #5 is junk, Ruil's clone is not good. 

The above was done by Ruili 

= Repeat =

The following exp was done by Paul 

== 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 ==

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

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

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

4: pLentiPlus, #3, EcoR1+Xho1+Nde1 

5: pLVGP, EcoR1+Xho1 

6: pLentiPlus, EcoR1+Xho1 

7: Cre PCR fragment, EcoR1+Xho1 

8: Cre PCR fragment,, EcoR1+Xho1 

Purify vectors #5 & #6

Purify PCR fragment, #7 & #8

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

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

 

[[Image:IMG 1083.JPG]] 

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

 

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

 

[[Image:242+300.JPG]] 1: Marker 

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

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

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

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

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

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

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

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

 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

3: plentiCre(ID, 241) ,Xhoi+Nco1 

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

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

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

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

 Digestion and gel

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[[Category:Cloning]]

PLentiGP-Cre - Revision history

Hylei at 17:59, 21 July 2011