1858 1868 fMMLV

Cloning of 1858pEC-fMMLV, 1868pT7A30-fMMLV

Purpose: Cloning of 1858pEC-fMMLV, 1868pT7A30-fMMLV

Date: 2013-10-17

Notebook:

Page:

Clone ID:1858,1868

Experimenters: Shanyi

Results

cloning

Date:2013-3-

PCR using MMLV DNA, clone 1713

MMLV-Nde1-F
atcgac CAT ATG ACCCTAAATATAGAAGATGAGC

MMLV-L139P-R cagtgtaccactggtgggacggtgg cgg cccgctcaagaggttg

MMLV-L139P-F caacctcttgagcggg ccg ccaccgtcccaccagtggtacactg
MMLV-D200N-R ctctgtgcagtgcctc att aaacagggtgggactg

MMLV-D200N-F cagtcccaccctgttt aat gaggcactgcacagag
MMLV-T330P-R caattaaacagagtccc cgg tttggtgagagggtac

MMLV-T330P-F gtaccctctcaccaaa ccg gggactctgtttaattg
MMLV-L603W-E607K-R gatctctttgcc ttt tgatgtgag cca cccacgccttctgtat

MMLV-L603W-E607K-F cagaaggcgtggg tgg ctcacatca aaa ggcaaagagatcaaa
MMLV-Xho1-R
(CTCCAGACACCTCTACCCTCCTC cat cac cat cac cat cac CTCGAG cgatca)
tgatcgCTCGAGgtgatggtgatggtgatgGAGGAGGGTAGAGGTGTCTGGAG

then use Fragment1,2,3 ,4,5as template MMLV-Nde1-F+MMLV-Xho1-R as primers

digest with NdeI+XhoI

Colony Screening

Date: 2013

DIGEST WITH  NdeI+XhoI

SEND 1858-2 TO SEQUENCE

cloning of 1868

subclone from1858-2

digest with NdeI+XhoI

Colony Screening

Date: 2013

DIGEST WITH  NdeI+XhoI

digest with BamHI

pick another 6 colonies

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