1284-pCMV5-Fc(3c)-NPR1-Cyto

Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Purpose: Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Date: 2012-7-27---2012-7-30

Notebook:0

Page:

Clone ID:1284

Experimenters: yingying

Results

cloning

PCR NPR1

tempate:kinase 7901 B07

using NPR1-Cyto-Nhe1-F catgac GCTAGC agtagccttgagaggcacctg NPR1-Xba1-R

	lane 6	lane 7
	PCR product	1262,pCMV5-Fc(3C)
enzymes	Nhe1+Xba1

Colony Screening

Date: 2012-7-30

Make miniprep and digest with Nhe1+Xba1

no insert

Result intepretation/Summary

• <br>

Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Purpose: Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Date: 2012-7-31--2012-8-2

Notebook:0

Page:

Clone ID:1284

Experimenters: yingying

Results

cloning

PCR NPR1

tempate:kinase 7901 B07

using NPR1-Cyto-Nhe1-F catgac GCTAGC agtagccttgagaggcacctg NPR1-Xba1-R

	lane 7	lane 8
	PCR product	1262,pCMV5-Fc(3C)
enzymes	Nhe1+Xba1

Colony Screening

Date: 2012-8-2

Make miniprep and digest with Nhe1+Xba1

no right clones

Result intepretation/Summary

• <br>

Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Purpose: Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Date: 2012-8-3---2012-8-6

Notebook:0

Page:

Clone ID:1284

Experimenters: yingying

Results

cloning

	band 1	band 2
	PCR product	1263-2,pCMV5-Fc(3c)-RIPK3
enzymes	Nhe1+Xba1

Colony Screening

Date: 2012-8-6

Make miniprep and digest with Nhe1+Xba1

no insert

Result intepretation/Summary

• <br>

Conclusion

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Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Purpose: Cloning of 1284-pCMV5-Fc(3c)-NPR1-Cyto

Date: 2012-8-7---2012-8-10

Notebook:0

Page:

Clone ID:1284

Experimenters: yingying

Results

cloning

PCR NPR1

tempate:kinase 7901 B07

using NPR1-Cyto-Nhe1-F catgac GCTAGC agtagccttgagaggcacctg NPR1-Not1-R

	lane 3	band
	PCR product	1263, pCMV5-Fc(3c)-RIPK3
enzymes	Nhe1+Not1

Colony Screening

Date: 2012-8-10

Make miniprep and digest with Nhe1+Not1

9/3/2012

Digest Maxiprep DNA with

Nhe1+ Not1--> GOI

Not1 + Xba1--> to see if SVLT is in the vector

1	2	3	4	5	6	7	8	9	10	11	12
1224	1263	1264	1269	1276	1284	1224	1263	1264	1269	1276	1284
Nhe1+ Not1	Nhe1+ Not1	Nhe1+ Not1	Nhe1+ Not1	Nhe1+ Not1	Nhe1+ Not1	Not1+ Xba1	Not1+ Xba1	Not1+ Xba1	Not1+ Xba1	Not1+ Xba1	Not1+ Xba1
		lot of RNA		lot of RNA

1224: vector with SVLT, digest with Not1 + Nhe1: why so many bands??

1263: why multiple bands with Nhe1 + Not1 digestion? there is an internal Xba1 site, digest with

Not1 + Xba1 --> 500 bp

Transfection: cell aggreggation: 1284 and 1276

Result intepretation/Summary

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Conclusion

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