1263-pCMV5-Fc(3c)-RIPK3

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Cloning of 1263-pCMV5-Fc(3c)-RIPK3

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Purpose: Cloning of 1263-pCMV5-Fc(3c)-RIPK3

Date: 2012--7-11----2012-7-11

Notebook:0

Page:

Clone ID:1263

Experimenters: yingying

Results

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cloning

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PCR using

template:kinase7899 A11
RIPK3-Nhe1-F
atcgacc gct agc ATGTCGTGCGTCAAGTTATGGCCCAG
RIPK3-Xho1-F wrong primer
gtgctagctcgaGCTATTTCCCGCTATGATTATACCAAC




   lane 1
   lane 2

 PCR product
 pCMV5-Fc(3C) 1262
enzymes
            Xho1 + Nhe1

Looks like the vector is not right. It should not have a small band


Colony Screening

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Result intepretation/Summary

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  • <br>

Conclusion

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Cloning of 1263-pCMV5-Fc(3c)-RIPK3

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Purpose: Cloning of 1263-pCMV5-Fc(3c)-RIPK3

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

Notebook:0

Page:

Clone ID:1263

Experimenters: yingying

Results

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cloning

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PCR using these primers
RIPK3-Nhe1-F
atcgacc gct agc ATGTCGTGCGTCAAGTTATGGCCCAG

RIPK3-Not1-R gatgcaagGCGGCCGCCTATTTCCCGCTATGATTATACCAAC

1263+1264+1269+1276+1284 clone 7-27-2012.JPG


 band 1
 band 2

 PCRproduct
 1262,pCMV5-Fc(3C)
enzymes
          Nhe1+  Not1



Colony Screening

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Date: 2012-7-30

Make miniprep and digest with Nhe1+ Not1


c

Result intepretation/Summary

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  • <br>

Conclusion

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Cloning of 1263-pCMV5-Fc(3c)-RIPK3

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Purpose: Cloning of 1263-pCMV5-Fc(3c)-RIPK3

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

Notebook:0

Page:

Clone ID:1263

Experimenters: yingying

Results

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cloning

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 band 1
 band 2

 PCRproduct
 1262,pCMV5-Fc(3C)
enzymes
           Nhe1+ Not1



Colony Screening

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Date: 2012-8-2

Make miniprep and digest with Nhe1+ Not1


digest detect again: EcoR1+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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