Editing 3009 pCMV5-Flag-H-IgG2b

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Date: 1/24/2018

Digest miniprep DNA with Xho1

vector: pMiniT2

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[[File:948-3135-3009-3001-950 digestion2.JPG.JPG|RTENOTITLE]]

1/25/2018

Miniprep DNA in pMiniT2 vector digested with Xho1

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Digest of 3009pMiniT

Enzymes:BsrGI+NheI

Time:1hour

Buffer:NEB2

[[File:3009pminitedigestion1.JPG|RTENOTITLE]]

'''Digest of 3009 pMiniT'''

Enzymes:XhoI+NheI

Time:30min

Buffer:Fastfigest

[[File:3009pminit2digestion.JPG|RTENOTITLE]]

'''Subclone of 945'''

1:1727 XbaI+NcoI 173&nbsp; Cutsmart

2:1834 XbaI+NcoI 173&nbsp; Cutsmart

3:945pMiniT XbaI+NcoI 1326&nbsp; Cutsmart

4:3009pMiniT BsrGI+NheI 371&nbsp; &nbsp;NEB 2

Time:30min

'''Result:1727 can see band as vector, 945pminiT2 can see band as insert, cut out and combine,low concentration of 309pMiniT'''

[[File:Subclone of 945.JPG|RTENOTITLE]

Date:3/15/2018

'''3009pMiniT2 digestion test <span style="font-size:larger;">1</span>'''

Restriction enzymes:BsrG I+NheI

Buffer:NEB 2

Time:30min

'''Result:seems Lane 4 has band'''

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'''3009pMIniT2 digestion test <span style="font-size:larger;">2</span>'''

Restriction enzymes:XhoI

Buffer:Fastdigest

Time:10min

7:3009-4pminiT2 (sequenced)

'''Result:some of them have bands, Lane 4 has band too.'''

[[File:3009pMIniT2 digestion test 2.JPG|RTENOTITLE]]

'''3009pMiniT2 digestion Test&nbsp;'''<span style="font-size:larger;">'''3'''</span>

1 3009-4 BsrGI +KpnI

2 3009#5 BsrGI +KpnI

3 3009#5 BsrGI +NdeI

4 3009#5 NdeI +NheI

Buffer:NEB 2

Time:30min

[[File:3009pMiniT2 digestion Test 3.JPG|RTENOTITLE]]



== Analysis of&nbsp;3009-4 in pMiniT2 ==

[[Category:Cloning]]3009-4 in pMiniT2 digested with Nhe1-BsrG1, Kpn1-BsrG1

Date: 3/26/18, by Paul

Buffer: FastDigest

37 oC 1.5 hour

[[File:3009 4 BsrG1 Kpn1 Nhe1.JPG]]

don't know if Nhe1 sitte is present, need to transform INV110