PLentiGP-Cre

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

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

1. Marker.

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

5. Digested Cre fragment with ECoRI+ XHoI

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)

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,
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

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

Run gel and purify PCR fragment.

--Paul.xu 16:26, 10 July 2011 (EST)

Cloning

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

--Paul.xu 16:08, 10 July 2011 (EST)

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.

--Paul.xu 16:08, 10 July 2011 (EST)

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;

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

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



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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