Crm at 18:12, 4 April 2018

2018-04-04T18:12:28Z

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'''PCR of 947'''

Lane1,2:Fragment 1 Primer:945 F+947 R

Lane3,4:Fragment 2 Primer:947 F+945 R

Buffer:Platinum HiFi supermix

'''Results:good, cut out and do next PCR'''

[[File:PCR of 947.JPG|RTENOTITLE]]

second PCR of 947

Primer: 945 F+945 R

Buffer:Patinum HiFi supermix

'''Results:good, cut out the first band '''

[[File:Second PCR of 947.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]]

'''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]]

== 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]]

== '''Date:3/15/2018''' ==

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

Buffer:cutsmart

Vecotor:1727

XbaI+NcoI

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

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

Date:3/19/2018

'''Miniprep of 947_947pMiniT2_3131_3152'''

947:1-4 XbaI+NcoI-HF 1394 Cutsmart

947pMiniT2:5-8 ECoRI Cutsmart

3131:9-12 BssHII+XhoI 342 Cutsmart

3152:13-16 BsrGI+KpnI 371 NEB 2

'''Result: sent lane 2, lane 16 to do sequence'''

[[File:IMG 3621.JPG|RTENOTITLE]]

Date:3/20/2018

'''PCR screening of 947'''

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

Lane 3,4:945-F;945-R

Length:1326bp

Extension time:1.5min

2X PCR Mix:Platinum SuperMix

[[File:IMG 3628.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
|-
| 
| BssHII
| 1856
| 4,5,6
|-
| 945
| BglII
| 628
| 11,12
|-
| 
| BssHII
| 1838
| 13
|-
| 3165
| SalI+BsrGI
| 693
| 8
|-
| 
| HindIII+BsrGI
| 441
| 10
|-
| 3166
| SalI+BsrGI
| 1234
| 9
|-
| 
| XbaI
| 2049
| 7
|-
| 
| BglII
| 3259
| to do
|}

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

== Date:4/2/2018 ==

'''redo Subclone of 947'''

Vector:1727

Insert:947pMiniT

Size:1344 bp

[[File:IMG 0608.JPG|RTENOTITLE]]

== Date:4/4/2018 ==

'''miniprep digestion of 984_947_3152'''

#Name Enzymes Size:

1-4:984 XbaI+NheI                     957bp

5 :982(973-1pMiniT) XbaI+NheI

6-9:947 XbaI+NcoI-HF                1344bp

10:947pMiniT XbaI+NcoI-HF

11:947(from PCR cloning) XbaI+NcoI-HF

12-15:3152 BsrGI+NheI              371bp

16: 3162(Vector) BsrGI+NheI

[[File:IMG 0615.JPG|RTENOTITLE]]
[[Category:Pages with broken file links]] [[Category:Cloning]]

947 pT7A30-6His-pMBP - Revision history

Crm at 18:12, 4 April 2018