Editing 945 pT7A30-pMBP

'''945 PCR'''

2X PCR Buffer:Platinum Supermix HiFi

Template:1281

Primer:945 F+ 945 R

'''Results:good, cut out and did gel purfication to prepare next step.'''

[[File:945PCR.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]]

'''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]

== 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

[[File:Miniprepof 945 3167.JPG|RTENOTITLE]]

'''PCR Screening of 945'''

Lane 1,2: pT7A3-seq-F;945R

Lane 3: 945 F; 947R

extension time:1.5min

2X PCR Mix:Platinum

length:1300 bp

[[File:IMG 3631.JPG|RTENOTITLE]]

Date:2/26/2018

'''Test of 947_945_3166_3165'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Name
| Enzymes
| Size (bp)
| Lane
|-
| 947
| BglII
| 646
| 1,2,3
|-
| <br/>
| BssHII
| 1856
| 4,5,6
|-
| 945
| BglII
| 628
| 11,12
|-
| <br/>
| BssHII
| 1838
| 13
|-
| 3165
| SalI+BsrGI
| 693
| 8
|-
| <br/>
| HindIII+BsrGI
| 441
| 10
|-
| 3166
| SalI+BsrGI
| 1234
| 9
|-
| <br/>
| XbaI
| 2049
| 7
|-
| <br/>
| BglII
| 3259
| to do
|}

[[File:IMG 36521.JPG|RTENOTITLE]]

'''945 subclone'''

'''Size:1326 bp'''

'''Enzymes:Xba1+Nco1'''

[[File:945subclone.JPG|RTENOTITLE]]

'''Miniprep digestion of 945_986_944_3127'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| #
| Lane
| Size
| Enzymes
| Note
|-
| 945
| 1-4
| 1326bp
| Xba1-Nco1<br/>
| subclone
|-
| 986
| 5-8
| 774bp
| HindIII+XhoI
| ~4kbp
|-
| 944
| 9-12
| 2334bp
| NdeI+XhoI
| PCR
|-
| 3127
| 13-16
| 402bp
| Nde1-HindIII<br/>
| Gibson Mix
|}



 [[File:IMG 0658.JPG|RTENOTITLE]]

[[Category:Cloning]]