1048-pEC-RNaseI

Cloning of 1048-pEC-RNaseI

Purpose: Cloning of1048-pEC-RNaseI

Date: 2013-3-

Notebook:

Page:

Clone ID:1048

Experimenters: Shanyi

Results

cloning

Date:2013-3-

gene synthesis

Fragment1: RnaseI-1 ~RnaseI-20, ~650 bp,
Fragment 2: RnaseI-21~40, ~740 bp,

 then use Fragment1+Fragment 2 as template

RnaseI-1+RnaseI-40 as primers

digest :

	band 2,3	band 4
	PCR product	pEC
enzymes	Nco1+ Xho1

Colony Screening

Date: 2013

send 1048-1 to sequence, has a G to T mutation and frame shift,  need to use use oligo to fix the frame shift and the mutation.

cloning

pcr

Fragment1: use RnaseI-Nco1-F and RnaseI-18 as primer
Fragment 2: use RnaseI-19 and RnaseI-40 as primer

then use Fragment1+Fragment 2 as template

RnaseI-1+RnaseI-40 as primers

digest :

	band 1	band3
	pEC	PCR product
enzymes	Nco1+ Xho1

Colony Screening

Date: 2013

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