1224-pCMV5-Fc(3C)-Dest-SVLT

Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

Purpose: Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

Date: 2012-7-6---2012-7-16

Notebook:0

Page:

Clone ID:1224

Experimenters: yingying

Results

subcloning

	band 1	band 2
	413 pCDNA3-YFP-IRES-SVLT	1222 pCMV5-Fc(3C)-Dest
enzymes	Not1 + Xba1

Colony Screening

Date: 2012-7-16

Make miniprep and digest with Not1 + Xba1

clone 3 may be right

clone 3 not the right clone

Result intepretation/Summary

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Conclusion

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Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

Purpose: Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

Date: 2012-7-17---2012-7-27

Notebook:0

Page:

Clone ID:1224

Experimenters: yingying

Results

subcloning

	band 1	band 2
	413 pCDNA3-YFP-IRES-SVLT	1222-3 pCMV5-Fc(3C)-Dest
enzymes	Not1 + Xba1

Colony Screening

Date: 2012-7-19

Make miniprep and digest with Not1 + Xba1

clone 1,2,3,4,5,6,7,8 may be right

digest detect again with :EcoR1+Xba1

2012-7-24

Gateway 393+1224 digest QC

Lane	1	2	3	4	5
plasmid	393 EGFP+1224pCMV5-Fc(3C)-Dest-SVLT				1224pCMV5-Fc(3C)-Dest-SVLT
enzymes	Nhe+Not

plasmid QC: 4 clonies above

no lane 

2012-7-27

Gateway 393+1224 digest detect :Nhe+Not

clone 3,4,7,8 no insert

2012-8-6

gateway reaction 393+1224-2

PCR screen 2012-8-7

Fc-Seq-F+IRES-R-seq

lane 1 :positive control 1224-2

Gateway reaction

2012-8-9

1--6 393+1224 6clones ;

7--1224;

8-13 393+1238 6 clones; clone8,10,11,12 transfected into cells,EGFP positive

14--1238;

1-6 393+456 6clones; clone1,2,3 transfected into cells,EGFP positive

7--456;

8--13 393+1191 6clones; clone8,9,10,11,12 transfected into cells,EGFP positive

14--1191

=

8/9/2012: 1224 + 393: Digest with Nhe1 + Not1

2012-8-10

393+1224 pcr sreenning of purified plasmid DNA: Fc-Seq-F+IRES-R-seq using Phusion (homemade)

lane 1-6: six clones;   lane7: 1224plasmid

8/10/2012 PCR screening of bacterial colonies using Fc-Seq-F+IRES-R-seq and Taq polymerase

c:393+1238

1224 digest detect:2012-8-14

Result intepretation/Summary

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Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

Purpose: Cloning of 1224-pCMV5-Fc(3C)-Dest-SVLT

Date: 2012-8-22---2012-8-23

Notebook:0

Page:

Clone ID:1224

Experimenters: yingying

Results

cloning

pick 6 clonies(2012.7.17) for growing

Colony Screening

Date: 2012-8-23

Make miniprep and digest with

clone 6 may be right

8/24/2012, test plasmid stability

Restransform ccdbsurvial with clone #6 plasmid DNA , pick 8 clones. digest miniprep

with Not1 + Xba1.

2012-8-28

1:Not1+Xba1

2-8:EcoR1+Xba1

Result intepretation/Summary

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Conclusion

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