ELN - User contributions [en]

File:1353 Digestion.JPG

2012-08-24T19:49:20Z

Jennifer.truong:

File:Vector Digestions.JPG

2012-08-24T19:33:26Z

Jennifer.truong:

File:1343 Digestions.JPG

2012-08-24T19:33:17Z

Jennifer.truong:

File:0824 Digestion.JPG

2012-08-24T19:16:13Z

Jennifer.truong:

File:1303 Digestion.JPG

2012-08-24T19:16:04Z

Jennifer.truong:

File:1303 Amp.JPG

2012-08-24T18:32:27Z

Jennifer.truong:

File:1344 Amp.JPG

2012-08-24T18:32:23Z

Jennifer.truong:

File:1341 Amp.JPG

2012-08-24T18:32:18Z

Jennifer.truong:

File:1342 Amp.JPG

2012-08-24T18:32:13Z

Jennifer.truong:

File:1343 Amp.JPG

2012-08-24T18:32:08Z

Jennifer.truong:

1329 pLVX-TX 05-HAwt-TagGFP

2012-08-24T18:15:06Z

Jennifer.truong:

== '''Cloning of 1329 pLVX-TX 05-HAwt-TagGFP''' ==

'''Purpose:''' Cloning of 1329 pLVX-TX 05-HAwt-TagGFP

'''Date: '''August 21, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1329

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 21, 2012

[[Image:0821 PCR Amp.JPG]]

See lane 1

PCR using these two oligos TX_05-HAwt-Xho1-F ctgaca CTC GAG gcc acc atgaaggcaatactagtagt tc

TX_05-HAwt-EcoR1-R (ctacagtgtagaatatgtatt CGA ATT CTG CAG T) ACTGCAGAATTCGaatacatattctacactgtag

Template 59 pHW2000-TX/05 HAwt [[Image:1329 Digestion.JPG]] 

Digested insert (lane 1) and vector 110 (lane 2) with RE Xho1 and EcoR1

Ligate together and transform

=== Colony Screening ===

Date: August 24, 2012 

Digested MiniPrep with RE Xho1 and EcoR1 

[[Image:1329_Screening.JPG]]

Sent sample 1 to sequencing

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

File:1329 Screening.JPG

2012-08-24T18:13:58Z

Jennifer.truong:

1330 pLVX-TX 05 -H-100P-TagGFP

2012-08-24T18:05:49Z

Jennifer.truong:

== '''Cloning of 1330 pLVX-TX 05 -H-100P-TagGFP''' ==

'''Purpose:''' Cloning of 1330 pLVX-TX 05 -H-100P-TagGFP

'''Date: '''August 21, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1330

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 21, 2012

[[Image:0821 PCR Amp.JPG]]

 

See lane 2

PCR using these two oligos TX_05-HAwt-Xho1-F ctgaca CTC GAG gcc acc atgaaggcaatactagtagt tc

TX_05-HAwt-EcoR1-R (ctacagtgtagaatatgtatt CGA ATT CTG CAG T) ACTGCAGAATTCGaatacatattctacactgtag

Template 58 pHW2000-TX/05 HA100P 

[[Image:1330 Digestion.JPG]] 

Digested insert (lane 3) and vector 110 (lane 4) with RE Xho1 and EcoR1

Ligate together and transform 

=== Colony Screening ===

Date: August 24, 2012

Digested MiniPrep with RE Xho1 and EcoR1 

[[Image:1330_Screening.JPG]]

Sent sample 1 to sequencing

 

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1332 pLVX-PR8-HA100S-TagGFP

2012-08-24T18:04:27Z

Jennifer.truong:

== '''Cloning of 1332 pLVX-PR8-HA100S-TagGFP''' ==

'''Purpose:''' Cloning of 1332 pLVX-PR8-HA100S-TagGFP

'''Date: '''August 21, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1331

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 21, 2012

[[Image:0821 PCR Amp.JPG]]

See lane 4

Cloning Method

PCR using these primers

PR8-HAwt-Sal1-F agctacGTCGACgccaccatgaaggcaaacctactggtc PR8-HAwt-BamH1-R (ctttgcagtgcagaatatgcatcgaggatcctcgaca) tgtcgaggatcctcgatgcatattctgcactgcaaag template: 56 pHW2000-PR8 HA100S

Digest PCR product with Sal1 + BamH1 Digest vector with Xho1 + BamH1 

[[Image:1332 Digestion.JPG]] 

Digested insert (lane 7) with RE Sal1 and BamH1, digested vector 110 with RE Xho1 and BamH1 (lane 8) 

LIgate together and transform 

=== Colony Screening ===

Date: August 24, 2012

Digested MiniPrep with RE Xho1 and BamH1 

[[Image:1332_Screening.JPG]]

Sent sample 4 to sequencing 

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1333 pLVX-CA 04-HAwt-TagGFP

2012-08-24T18:03:39Z

Jennifer.truong:

== '''Cloning of 1333 pLVX-CA 04-HAwt-TagGFP''' ==

'''Purpose:''' Cloning of 1333 pLVX-CA 04-HAwt-TagGFP

'''Date: '''August 21, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1333

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 21, 2012

[[Image:0821 PCR Amp.JPG]]

 

See lane 5

PCR using these two oligos TX_05-HAwt-Xho1-F ctgaca CTC GAG gcc acc atgaaggcaatactagtagt tc

TX_05-HAwt-EcoR-R (ctacagtgtagaatatgtatt CGA ATT CTG CAG T) ACTGCAGAATTCGaatacatattctacactgtag template : 55 pHW2000-CA/04 HAwt 

[[Image:1333 Digestion.JPG]]

Digested insert (lane 9) and vector 110 (lane 10) with RE Xho1 and EcoR1

Ligate together and transform

=== Colony Screening ===

Date: August 24, 2012

Digested MiniPrep with RE Xho1 and EcoR1 

[[Image:1333_Screening.JPG]]

Sent sample 1 to sequencing

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1334 pLVX-CA 04-HA100S-TagGFP

2012-08-24T18:02:54Z

Jennifer.truong:

== '''Cloning of 1334 pLVX-CA 04-HA100S-TagGFP''' ==

'''Purpose:''' Cloning of 1334 pLVX-CA 04-HA100S-TagGFP

'''Date: '''August 21, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1334

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 21, 2012

[[Image:0821 PCR Amp.JPG]]

See lane 6

PCR using these two oligos TX_05-HAwt-Xho1-F ctgaca CTC GAG gcc acc atgaaggcaatactagtagt tc

TX_05-HAwt-EcoR-R (ctacagtgtagaatatgtatt CGA ATT CTG CAG T) ACTGCAGAATTCGaatacatattctacactgtag template: 54 pHW2000-CA/04 HA100S 

[[Image:1334 Digestion.JPG]] 

Digested insert (lane 11) and vector 110 (lane 12) with RE Xho1 and EcoR1

Ligate together and transform 

=== Colony Screening ===

Date: August 24, 2012

Digested MiniPrep with RE Xho1 and EcoR1 

[[Image:1334_Screening.JPG]]

Sent sample 1 to sequencing

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

File:1330 Screening.JPG

2012-08-24T17:57:13Z

Jennifer.truong:

File:1331 Screening.JPG

2012-08-24T17:57:08Z

Jennifer.truong:

File:1332 Screening.JPG

2012-08-24T17:57:03Z

Jennifer.truong:

File:1333 Screening.JPG

2012-08-24T17:56:56Z

Jennifer.truong:

File:1334 Screening.JPG

2012-08-24T17:56:51Z

Jennifer.truong:

File:1335 Screening.JPG

2012-08-24T17:56:47Z

Jennifer.truong:

File:115 Screening.JPG

2012-08-24T17:56:42Z

Jennifer.truong:

File:1335 Subcloning.JPG

2012-08-24T17:28:22Z

Jennifer.truong: uploaded a new version of "File:1335 Subcloning.JPG"

File:1335 Insert.JPG

2012-08-24T17:28:12Z

Jennifer.truong:

File:1287 Digestion 2.JPG

2012-08-24T17:00:33Z

Jennifer.truong:

File:1346 Screening.JPG

2012-08-24T15:51:22Z

Jennifer.truong:

File:1287 Screening.JPG

2012-08-24T15:51:17Z

Jennifer.truong:

File:1329 Digestion.JPG

2012-08-21T20:52:14Z

Jennifer.truong:

File:1330 Digestion.JPG

2012-08-21T20:52:10Z

Jennifer.truong:

File:1331 Digestion.JPG

2012-08-21T20:52:03Z

Jennifer.truong:

File:1332 Digestion.JPG

2012-08-21T20:51:58Z

Jennifer.truong:

File:1333 Digestion.JPG

2012-08-21T20:51:53Z

Jennifer.truong:

File:1334 Digestion.JPG

2012-08-21T20:51:48Z

Jennifer.truong:

File:1335 Digestion.JPG

2012-08-21T20:51:43Z

Jennifer.truong:

File:1335 Amp.JPG

2012-08-21T18:32:42Z

Jennifer.truong: uploaded a new version of "File:1335 Amp.JPG"

File:0821 PCR Amp.JPG

2012-08-21T18:09:09Z

Jennifer.truong:

File:115 Digestion.JPG

2012-08-21T14:52:11Z

Jennifer.truong:

1297 pET-6H-VPMLK-TAT-NLS

2012-08-21T13:42:46Z

Jennifer.truong:

== '''Cloning of 1297 pET-6H-VPMLK-TAT-NLS''' ==

'''Purpose:''' Cloning of 1297 pET-6H-VPMLK-TAT-NLS

'''Date: '''August 13, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1297

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 13, 2012\

Gene synthesis

Purify PCR product, see lane 2, (113 bp, need to add 10% isopropanol becasue the fragment is very small).

[[Image:1297 0813 Amp.JPG]]

Digest PCR product with RE Nco1 + Nde1. Digest vector 1128 pDB.His.PfuMBP-(tev) with Nco1 + Nde1. Ligate together overnight and transform 

 

Date: August 14, 2012

Insert disappeared after digestion, need to reamplify, see lane 1

[[Image:0814 Amp.JPG]]

Date: August 15, 2012 

[[Image:1297 0815 Digestion.JPG]] 

Digest PCR product with RE Nco1 + Nde1 (lane 1) Digest vector pDB.His.MBP, V14 with Nco1 + Nde1 (lane 2) Ligate together and transform 

=== Colony Screening ===

Date: August 20, 2012 

[[Image:1297 Screening.JPG]]

Digested MiniPrep with RE Nco1 and Nde1

Faint band at expected size (~100 bp) maybe send to sequencing

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1217 pDB.His.PfuMBP-(3C)

2012-08-21T13:15:21Z

Jennifer.truong:

== '''Cloning of 1217 pDB.His.PfuMBP-(3C)''' ==

'''Purpose:''' Cloning of 1217 pDB.His.PfuMBP-(3C)

'''Date: '''June 26, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: 1217'''

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

[[Image:0626 Amp.JPG]] 

Digested pDB.His.PfuMBP 1129 with Nde1 + BamH1 (see lane 1), looks like it is not digested. Ran out of 1129 vector, growing new from gycerol stock. Will redo tomorrow. Stored annealed oligos in box. 

Date: June 27, 2012 

Digested pDB.His.PfuMBP 1129 with Nde1 + BamH1 (see lane 1) again, same 2 bands happened. 

[[Image:0627 Digestion.JPG]]

Digested vector separately with BamHI and Nde1, digest with NEB enzymes 

[[Image:1192 digestion.JPG]]

Digested with NEB BamHI and Nde1

[[Image:1217 vector.JPG]]

Date: August 17, 2012 

Digested pDB.His.PfuMBP 1129 with Nde1 + BamH1 (see lane 1) 

[[Image:1217_Vector_Digestion.JPG]]

=== Colony Screening ===

[[Image:1217 screening.JPG]] 

Digested with RE Pst + Xba1 

Very light band at expected 1383 bp, very unclear 

Sequencing result is not good, need to do it again. 

Date: August 20, 2012

[[Image:1217 0820 Screening.JPG]]

Digest with Pst + Xba1--->get a band of 1272 bp Sent sample 5 to sequencing 

Clones 1, 2, 3, 5 may be correct

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1207-pDB.His.MBP-(3C)

2012-08-21T13:08:12Z

Jennifer.truong:

== '''Cloning of 1207-pDB.His.MBP-(3C)''' ==

'''Purpose:''' Cloning of 1207-pDB.His.MBP-(3C)

'''Date:&nbsp;''' 2012-6-29----

'''Notebook:'''

'''Page:'''

'''Clone ID: 1207'''

'''Experimenters:'''yingying lu

== '''Cloning''' ==

[[Image:1207 clone 6-29-2012.JPG]] 

lane 1: digest 360 pDB.His.MBP with Nde1 + BamH1, ligate to annealled oligos MBP-3C-T T Agc CTG GAA GTT CTG TTC CAG GGG CCC CAT ATG CTG CAG gct agc g MBP-3C-B gatccgctagcCTGCAGCATATGGGGCCCCTGGAACAGAACTTCCAGgc 

Date: August 17, 2012 

lane 1: digest 360 pDB.His.MBP with Nde1 + BamH1, ligate to annealled oligos MBP-3C-T T Agc CTG GAA GTT CTG TTC CAG GGG CCC CAT ATG CTG CAG gct agc g MBP-3C-B gatccgctagcCTGCAGCATATGGGGCCCCTGGAACAGAACTTCCAGgc

[[Image:1207_Vector_Digestion.JPG]] 

 

 

== '''Colony Screening''' ==

Date: August 20, 2012 

[[Image:1207 0820 Screening.JPG]]

Digest with Pst + Xba1--->1.2 kb 

Clones 1, 2, 3, 4, 5 and 6 may be correct

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

1283 pDB.His.MBP.Ent

2012-08-20T20:16:18Z

Jennifer.truong:

== '''Cloning of 1283 pDB.His.MBP.Ent''' ==

'''Purpose:''' Cloning of 1283 pDB.His.MBP.Ent

'''Date: '''July 31, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''1283

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: July 31, 2012 

[[Image:0731 Amp.JPG]] 

PCR MBP from pMAL-p5E using 1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc 1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC 1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG

Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 

See lane 2, expected size ~1400 bp 

Cut out band and digested 

[[Image:1283 Digestion.JPG]] 

Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2)  

Cut out bands, purify and ligate together overnight

Date: August 7, 2012 

[[Image:1283 0807 Amp.JPG]] 

Sequencing came back contaminated, started all over 

See lanes 1 and 2 

PCR MBP from pMAL-p5E using 1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc 1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC 1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG

Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14

See lane 2, expected size ~1400 bp

Cut out band and digested 

[[Image:1283 0810 Digestion.JPG]] 

Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2)

Cut out bands, purify and ligate together overnight 

Date: August 9, 2012

Digestion was messed up from earlier because of the gel, started all over

[[Image:1283 0809 Amp.JPG]]

Cut out lanes 1 and 2 for digestion, save 3 and 4 in box just incase something happens

Date: August 17, 2012 

[[Image:1283 0817 Digestion.JPG]] 

Digest PCR product with Xba1 + Nde1 (lane 7), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 8)

Cut out bands, purify and ligate together overnight 

=== Colony Screening ===

Date: August 3, 2012 

[[Image:1283 Screening.JPG]] 

Digested MiniPrep plasmids with RE Xba1 + Nde1 in lanes 1 - 6 (samples 1-6 respectively) 

Digested sample 1 with Xba1 + Nco1 in lane 8, and digested sample 1 with Xba1 + Kpn1 in lane 9

All correct sizes, send sample 1 to sequencing

Clones 

'''These are contaminated clones'''
Date:  2012

[[Image:1283_0820_Screening.JPG]]

Clones 2, 3, 4, 5 and 6 may be correct

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

File:1207 0820 Screening.JPG

2012-08-20T20:04:18Z

Jennifer.truong:

File:1217 0820 Screening.JPG

2012-08-20T20:04:13Z

Jennifer.truong:

File:1283 0820 Screening.JPG

2012-08-20T20:04:00Z

Jennifer.truong:

File:1292 0820 Screening.JPG

2012-08-20T20:03:51Z

Jennifer.truong:

File:1297 Screening.JPG

2012-08-20T20:03:45Z

Jennifer.truong:

File:1298 Screening.JPG

2012-08-20T20:03:40Z

Jennifer.truong:

110 pLVX-TagGFP

2012-08-17T20:52:22Z

Jennifer.truong:

110: pLVX-TagGFP 

8/11/2012 

1 to 6 digest with Nde1 + Xba1 

1* digest with Nde1 + Xho1 

[[Image:110 115 8 11 2012.JPG]] 

 

Nde1--CMV--Xho1--EcoR1--BamH1--TagGFP--Xba1

 

== '''Cloning of 110 pLVX-TagGFP''' ==

'''Purpose:''' Cloning of 110 pLVX-TagGFP

'''Date: '''August 15, 2012 

'''Notebook: '''

'''Page:'''

'''Clone ID: '''110

'''Experimenters: '''Jennifer Truong

== '''Results''' ==

=== Cloning ===

Date: August 15, 2012 

subcloning TO 153 pLVX-Plus digest 109 pET28b-TagGFP with BamH1 + Xba1 -->> TagRFP insert (lane 1) Digest vector 153 pLVX-Plus with BamH1 + Xba1 -->> vector 

[[Image:110 Insert Digestion.JPG]]

Lost vector, digest vector again with RE BamH1 and Xba1, ligate together and transform

=== Colony Screening ===

Date: August 17, 2012 

Digested MiniPrep with RE BamH1 and Xba1

[[Image:110 0817 Screening.JPG]]

Digested samples 1, 2, 3 in lanes 1, 2, 3 respectively with RE EcoR1 and Xba1

[[Image:0817_Digestion_2.JPG]]

== '''Result intepretation/Summary''' ==

 

== '''Conclusion''' ==

 

[[Category:Cloning]]

File:0817 Digestion 2.JPG

2012-08-17T20:51:40Z

Jennifer.truong: