Editing Yijia Li 3 12 2018

== Date:3/12/2018 ==

'''3010 miniprep:'''

Restriction enzymes: BamHI+KpnI

Time:15min

Buffer:Fastdigest

'''Result:good, sent to do sequencing.#4'''

[[File:3010miniprep.JPG|RTENOTITLE]]

'''Miniprep of 945pMiniT2_947pMiniT2_3131_1727_1834'''

945pMiniT2:1-4 EcoRI

947pMiniT2:5-8 n EcoRI

3131:9-13 BssHII+XhoI

1727:14 XbaI+NcoI

1834:15 XbaI+NcoI

Buffer:Fastdigest

Time:30Min

'''Results:945pMiniT2 and 947pMiniT2 are good, so sent them to do sequencing. 3131 is not good, 1727 has no band, 1834 has one band.'''

[[File:Miniprep of 945 947 1727 1834.JPG|RTENOTITLE]]

'''Miniprep of 3168_3132_3161'''

3168:1-4 BsrGI+NheI

3132:5-8 BsrGI+NheI

3161:9-12 NdeI+XhoI

Buffer:Fastdigest

Time:30Min

'''Result:#7 seems good, so sent to do sequencing, the rest of them are suspicious'''

[[File:Miniprep of 3168 3132 3161.JPG|RTENOTITLE]]

'''Subclone of 3168'''

Retsriction enzymes:BsrGI+NheI

Time:30MIn

Buffer:Fastdigest

'''Results:good, combine and do the step.'''

[[File:Subclone of new3168.JPG|RTENOTITLE]]

'''Subclone of 3131:'''

Restriction enzymnes:BssHII+XhoI

Time:30min

Buffer:Fastdigest

'''result:good, combine and do ligate'''

[[File:Subclone of new 3131.JPG|RTENOTITLE]]

== Date:3/13/2018 ==

'''Test of 3165miniprep'''

Digest enzyme:BsrGI

Time:30min

Buffer:fastdigest

'''Results:not good, got three bands'''

[[File:Test of 3165.JPG|RTENOTITLE]]

'''Repeat subclone of 3152'''

Restriction enzymes:BsrGI+NheI

Buffer:NEB2

Time:30min

'''Result:Vector digest is good, but insert is not good.'''

[[File:Repeat subclone of 3152.JPG|RTENOTITLE]]

'''Test of 3165 miniprep digestion'''

1:SalI+BsrGI NEB2

2:SalI+KpnI Fastdigest

3:BsrGI NEB2

Time:30min

[[File:Test3165.JPG|RTENOTITLE]]

'''Digest of Vector 3130'''

made for Gibson mix

BamHI+KpnI

'''result:good, cut out and purify'''

[[File:Gibsonmix of 3130digestion.JPG|RTENOTITLE]]

'''PCR of 947'''

1281 as template

F1:945F+947R

F2:947F+945R

Extension time:1.5min

Annealling:55oC

Cycles:25

Volumn:25ul

2XPCR MIx:Platinum mix HiFi SUpermix

'''Results:good,combine F1+F2 and do the next step'''

[[File:PCR of 947F1+F2.JPG|RTENOTITLE]]

'''Digest of 3009pMiniT'''

Enzymes:BsrGI+NheI

Time:1hour

Buffer:NEB2

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

'''Digest of 30090pMiniT'''

Enzymes:XhoI+NheI

Time:30min

Buffer:Fastfigest

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

== Date:3/14/2018 ==

'''947 F3 PCR'''

F3:1,2

Negative Control:3

PCR Mix:Platinum

Primers:945F+945R

'''Result:Have bands, cut the top one and do the next step.'''

[[File:947F3PCR.JPG|RTENOTITLE]]

'''Miniprep digestion_3161_3168'''

3161:1,2 NdeI+XhoI Buffer:Cutsmart

3168:3 BsrGI+NheI

Buffer:NEB 2

Time:40min

'''Result:seems good, do sequencing'''

[[File:Miniprep of 3161 3168.JPG|RTENOTITLE]]

'''Miniprep digestion of 3131_3161_3167'''

Name length Enzymen Buffer

3131:1-4 342 BssHII+XhoI Cutsmart

3161:5-8 225 NdeI+XhoI Cutsmart

3167:9-12 397 BsrGI+NheI NEB 2

Time:40min

[[File:Miniprepof 3131 3167 3161.JPG|RTENOTITLE]]

'''Redo miniprep of 3131_3161_3167'''

3131:1-4 BssHII+XhoI 342 Cutsmart

3161:5-8 NdeI+XhoI 225 Cutsmart

3167:9-12 BsrGI+NheI 397 NEB 2

Time:40min

[[File:Redo miniprep of 3131 3161 3167.JPG|RTENOTITLE]]

'''miniprep digestion 3021'''

3021pMiniT2:BssHII+XbaI 374

3021:BssHII+XbaI 374 Cutsmart

3165:SalI+BsrGI 702 NEB 3

Time:30min

[[File:Redominiprep diegstion 3021.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'''

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

'''Redo Subclone of 3167'''

Vector:3163pCEH0

Insert:3027pMiniT2

BsrGI+KpnI

Buffer:NEB 2

Time:30min

'''Result:Cut out and combine'''

[[File:Redo Subclone 3167.JPG|RTENOTITLE]]



== Date:3/15/2018 ==

Gibson mix practice: 3001

miniprep digestion: BamHI+KpnI

Time:15moin

Buffer:Fastdigest

[[File:Gibsonmix 3001.JPG|RTENOTITLE]]

'''MiniPrep digestion of 3131'''

Enzymes:BssHII+XhoI

Buffer:Cutsmart

Time:40min

'''Result:2,3,4,seems good, do sequencing,select #3'''

[[File:Miniprepdigstion3131.JPG|RTENOTITLE]]

'''3009pMiniT2 digestion as Insert'''

'''Result:suspicious,Redo'''

[[File:3009pminit2digestion3.JPG|RTENOTITLE]]

'''Subclone of 947 Vector digstion''' Time:30min

Buffer:cutsmart

Vecotor:1727

XbaI+NcoI

'''Result:good, cut out and purify'''

[[File:947subclonevector.JPG|RTENOTITLE]]

'''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'''

[[File:IMG 3602.JPG|RTENOTITLE]]

'''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]]

'''Redo 3165 PCR 1st'''

F1:3028 as template

Primer:3165F1+3165R1

length440bp

extenstion time:1:30

F2:3129 as template

Primer:3165F2+3165R2

Buffer:Platinum

length:165bp

Extension time:15bp

[[File:3165PCRF1+F2.JPG|RTENOTITLE]]

'''Redo3166 PCR'''

Primer:3166F+3166R

Template:3017pCEL0

buffer:Platinum

length:60bp

extension time:15s

[[File:3166PCR.JPG|RTENOTITLE]]

== Date:3/16/2018 ==

'''Miniprep of 945_3167'''

945:1-4 Xba+NcoI Cutsmart 1326

3167:5-8 BsrGI+NheI NEB 2 397

Time:50min

Result:

[[File:Miniprepof 945 3167.JPG|RTENOTITLE]]
[[Category:My ELN]]