3257 pCMV5-anti-mkappa-rFc-His

Cloning of 3257 pCMV5-anti-mkappa-rFc-His

Purpose: Cloning of 3257 pCMV5-anti-mkappa-rFc-His

Date: 08, 15, 2018

Notebook: 

Page:

Clone ID: 3257

Experimenters: yijia

Results

Cloning

gene synthesis of 3257-F1

template:

Gene synthesis

3257-1TGTCACGAATTCACAGGTGCAGCTGGTG

3257-2GTCTCAGAGAGCCGCCAGGTTGAACCCATCCTCCGCCAGATTCCACCAGCTGCACCTGTG

3257-3TGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTTAGCGACACAGCCAT

3257-4CCCATTCTCTTCCCTTGCCAGGGGCCTGTCGGACCCACATCATGGCTGTGTCGCTAAAGG

3257-5GGCAAGGGAAGAGAATGGGTTGCCGCCATCGATACCGGCGGAGGCTACACCTACTACGCC

3257-6GCGTTGTCCCGGCTGATGGTGAATCTGCCCTTCACAGAATCGGCGTAGTAGGTGTAGCCT

3257-7TCAGCCGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAGCCTGAGG

3257-8AGTTGCCGCTGTAGGTCTTGGCGCAGTAGTACCGGGCTGTGTCCTCAGGCTTCAGGCTGT

3257-9AGACCTACAGCGGCAACTACTACAGCAACTACACCGTGGCCAACTACGGCACCACAGGCA

3257-10GGTACCGCTAGCGCTAGACACTGTGACCAGTGTTCCTCTGCCTGTGGTGCCGTAG

1st PCR of 3257-F2

PCR  from  1413 pCDNA3.4-Rabbit-IgG Heavy Chain
3257-F2 CACTGGTCACAGTGTCTAGCGCTAGCGGTACCAGCAAGCCCACGTGCCCAC

3257-Not1-R3  TTGATTGCGGCCGCTCAGCAGTGATGGTGATGATGATGTTTACCCGGAGAGCGGGAGATG

2nd PCR of 3257

combine both gene synthesis fragments and rabbit Fc fragment

Purify with column PCR again using

3257-1 TGTCACGAATTCACAGGTGCAGCTGGTG

3257-Not1-R3 TTGATTGCGGCCGCTCAGCAGTGATGGTGATGATGATGTTTACCCGGAGAGCGGGAGATG

1160bp

Vector digestion

Vector is 3075

 Colony screen

3257 PCR Screening

Miniprep of 3257

#	Lane	Enzymes	Length	Buffer	Note
3257	1-4(#1-4)	EcoR1+Not1	1160bp	Fastdigest	#2 to do sequence
vector control	3075	EcoR1+Not1	681bp	Fastddigest

10/16/2018: in order to digest with Nhe1, plasmid has to be isolated from INV110.

Conclusion

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