ELN - User contributions [en]

File:Cre taq pka qc 2 22 2012.jpg

2012-03-06T14:35:03Z

Yingying.lu:

File:Cre+taq+pka copy.jpg

2012-03-06T14:22:17Z

Yingying.lu:

Lentivirus QC 1-31-2012

2012-02-09T21:19:31Z

Yingying.lu:

Notebook: Xiaojing Wang

Page: 004171

 

493 pLVXPlus2-App-IRES-mCherry

528 pLVX-IRES-EGFP

220 pLenti-mKate2

 

493 528 220 lentivirus production-293T in 6 well plate

Use psPAX2 (503) 1 ÎŒg + VSVG (505) 0.5 ÎŒg + DNA 1.7 ÎŒg/0.85 ÎŒg 

2 days after transfection, take out 0.5 ml medium and add to 1 well of 293T cells growing on 12 well plate (50% confuluency). Add 1000X polybrene, so the final concentration is 1X .

Incubate for 4 days, take pictures. 

[[Image:493 528 220 lentivirus production 1.25.2012.jpg]] 

 

[[Category:Lentivirus]]

File:493 528 220 lentivirus production 1.25.2012.jpg

2012-02-09T21:19:06Z

Yingying.lu:

Infect Lentivirus 1-31-2012

2012-02-09T21:11:14Z

Yingying.lu: Blanked the page

252-pLenti-IRES-TFR-EGFP

2012-02-07T15:40:50Z

Yingying.lu:

Cloning of pLenti-IRES-TFR-EGFP (252)

Purpose: to clone pLenti-IRES-TFR-EGFP by Gene Synthesis

Date: 2012-1-24 to 2012-1-27

Notebook: Xiaojing Wang

Page: 004143---004144

Clone ID: 252

Experimenters: Xiaojing Wang

Results 

[[Image:252 PCR 1.24.2012 S.JPG]] 

digest: 

[[Image:252 digest 1.24.2012 S.JPG]][[Image:252's vector 1.24.2012 s.JPG]] 

digest PCR product and vector with Xho1 + Mlu1, run gel

the left band shows 252 insert, the right picture shows vector (pLentiPlus, 152)

gel purify, ligation, transformation

Colony Screening 

 

[[Image:252 digest 1.27.2012 s.JPG]] 

 

Select 5 clones to mini-prep, digested by Xho1 and Mlu1. 252-2,-3,-4,-5 may be correct. Control is 252-1.

Select 252-2 to sequence. 

 

[[Category:Cloning]]

File:252 digest 1.27.2012 s.JPG

2012-02-07T14:19:06Z

Yingying.lu:

File:252's vector 1.24.2012 s.JPG

2012-02-07T14:18:04Z

Yingying.lu:

File:252 digest 1.24.2012 S.JPG

2012-02-07T14:17:23Z

Yingying.lu:

File:252 PCR 1.24.2012 S.JPG

2012-02-07T14:16:11Z

Yingying.lu:

524-pCDNA3.1-E1A

2012-01-24T15:55:50Z

Yingying.lu: Created page with "Cloning of pCDNA3.1-E1A (524) Purpose: to clone pCDNA3.1-E1A by Gene Synthesis Date: 2012-1-18 to 2012-1-23 Notebook: Xiaojing Wang Page: 004163---004164 Clone ID: 524 Expe..."

Cloning of pCDNA3.1-E1A (524)

Purpose: to clone pCDNA3.1-E1A by Gene Synthesis

Date: 2012-1-18 to 2012-1-23

Notebook: Xiaojing Wang

Page: 004163---004164

Clone ID: 524

Experimenters: Xiaojing Wang

Results 

[[Image:524 PCR 1.6.2012S.JPG|114x296px]] 

digest: 

[[Image:521 524 vector digest 1.18.2012s.JPG]] 

digest PCR product and vector with EcoR1 +Xho1,run gel

the left band shows 524 insert, the right picture shows 335 vector (pCDNA3.1, 335)

gel purify, ligation, transformation

 Colony Screening 

[[Image:524 digest 1.23.2012.JPG]] 

Select 5 clones to mini-prep, digestd by EcoR1 and Xho1. 524-1,-3,-4,-5 may be correct. 

Select 524-1 to sequence. 

 

 

[[Category:Cloning]]

File:524 digest 1.23.2012.JPG

2012-01-24T15:54:38Z

Yingying.lu:

File:521 524 vector digest 1.18.2012s.JPG

2012-01-24T15:53:37Z

Yingying.lu: uploaded a new version of "File:521 524 vector digest 1.18.2012s.JPG"

File:524 PCR 1.6.2012S.JPG

2012-01-24T15:52:38Z

Yingying.lu:

File:523 digest 1.23.2012.JPG

2012-01-24T15:48:03Z

Yingying.lu:

File:523 and vector digest 1.19.2012.JPG

2012-01-24T15:46:47Z

Yingying.lu:

File:523 PCR 1.20.2012 S.JPG

2012-01-24T15:45:45Z

Yingying.lu:

522-pCDNA3.1-Bcl-X

2012-01-24T15:37:27Z

Yingying.lu:

Cloning of pCDNA3.1-Bcl-X (522)

Purpose: to clone pCDNA3.1-Bcl-X by Gene Synthesis

Date: 2012-1-18 to 2012-1-23

Notebook: Xiaojing Wang

Page: 004163---004164

Clone ID: 522

Experimenters: Xiaojing Wang

Results 

[[Image:522 PCR 1.18.2012.JPG|111x254px|522 PCR 1.18.2012.JPG]] 

digest: 

[[Image:522 and vector digest 1.19.2012.JPG]] 

digest PCR product and vector with Nhe1 +Xho1,run gel

the left band shows 522 insert, the right band shows 335 vector (pCDNA3.1, 335)

gel purify, ligation, transformation

 Colony Screening 

[[Image:522 digest 1.23.2012 s.JPG]] 

Select 5 clones to mini-prep, digestd by Nhe1 and Xho1. 522-2, -3, -5 may be correct. Control is 522-1.

Select 522-3 to sequence. 

 

 

[[Category:Cloning]]

File:522 digest 1.23.2012 s.JPG

2012-01-24T15:33:49Z

Yingying.lu:

521-pCDNA3.1-E1B-19K

2012-01-24T15:32:38Z

Yingying.lu:

Cloning of pCDNA3.1-E1B-19K (521)

Purpose: to clone pCDNA3.1-E1B-19K by Gene Synthesis

Date: 2012-1-18 to 2012-1-23

Notebook: Xiaojing Wang

Page: 004163---004164

Clone ID: 521

Experimenters: Xiaojing Wang

Results 

[[Image:521 PCR 1.6.2012.JPG|109x317px|521 PCR 1.6.2012.JPG]] 

digest: 

[[Image:521 524 vector digest 1.18.2012s.JPG]] 

digest PCR product and vector with EcoR1 +Xho1,run gel

the left band shows 521 insert, the right picture shows 335 vector (pCDNA3.1, 335)

gel purify, ligation, transformation

 

Colony Screening 

[[Image:521 digest 1.23.2012.JPG]] 

Select 5 clones to mini-prep, digestd by EcoR1 and Xho1. 521-1,-3,-4,-5 may be correct. Control is 521-1. 

Select 521-1 to sequence. 

 

[[Category:Cloning]]

File:522 and vector digest 1.19.2012.JPG

2012-01-24T15:31:31Z

Yingying.lu:

File:522 PCR 1.18.2012.JPG

2012-01-24T15:30:31Z

Yingying.lu:

File:521 digest 1.23.2012.JPG

2012-01-24T15:23:31Z

Yingying.lu:

File:521 PCR 1.6.2012.JPG

2012-01-24T15:16:50Z

Yingying.lu:

File:Day4 plate4 1.13.2012.JPG

2012-01-13T14:19:30Z

Yingying.lu: uploaded a new version of "File:Day4 plate4 1.13.2012.JPG"

File:Day4 plate3 1.13.2012.JPG

2012-01-13T14:13:01Z

Yingying.lu:

File:Day4 plate2 1.13.2012.JPG

2012-01-13T14:06:40Z

Yingying.lu:

File:Day4 plate1 1.13.2012.JPG

2012-01-13T14:04:33Z

Yingying.lu:

File:Day 3 1.13.2012.JPG

2012-01-13T14:01:48Z

Yingying.lu:

File:Day 2 1.12.2012.JPG

2012-01-12T16:43:43Z

Yingying.lu:

File:24h 1.11.2012.jpg

2012-01-12T16:41:06Z

Yingying.lu:

File:Day2 2 weeks 1.11.2012.jpg

2012-01-12T16:22:05Z

Yingying.lu:

File:Day3 plate4 1.12.2012s.JPG

2012-01-12T14:34:46Z

Yingying.lu:

File:Day3 plate3 1.12.2012s.JPG

2012-01-12T14:34:07Z

Yingying.lu:

File:Day3 plate2 1.12.2012s.JPG

2012-01-12T14:33:27Z

Yingying.lu:

File:Day3 plate1 1.12.2012s.JPG

2012-01-12T14:32:37Z

Yingying.lu:

File:Day3 2 weeks 1.12.2012.jpg

2012-01-12T14:27:01Z

Yingying.lu:

File:Day1 2 weeks 1.10.2012.jpg

2012-01-12T14:26:27Z

Yingying.lu:

File:24h 2 weeks 1.10.2012.jpg

2012-01-12T14:25:17Z

Yingying.lu:

File:24h plate4 blue 1.10.2012.jpg

2012-01-10T20:50:24Z

Yingying.lu:

File:24h plate3 blue 1.10.2012.jpg

2012-01-10T20:49:53Z

Yingying.lu:

File:24h plate2 blue 1.10.2012.jpg

2012-01-10T20:49:21Z

Yingying.lu:

File:24h plate4 1.10.2012.jpg

2012-01-10T20:48:47Z

Yingying.lu:

File:24h plate3 1.10.2012.jpg

2012-01-10T20:48:11Z

Yingying.lu:

File:24h plate2 1.10.2012.jpg

2012-01-10T20:47:36Z

Yingying.lu:

File:24h plate1 blue 1.10.2012.jpg

2012-01-10T20:46:13Z

Yingying.lu:

File:24h plate1 1.10.2012.jpg

2012-01-10T20:45:19Z

Yingying.lu:

519-pAD5.CAG-mMKOS

2012-01-10T18:06:11Z

Yingying.lu:

Cloning ofpAD5.CAG-mMKOS (519) Purpose: to clone pAD5.CAG-mMKOS by subclone Date: 2011-12-30 to 2012-1-5 Notebook: Xiaojing Wang Page: 004147---004147 Clone ID: 519 Experimenters: Xiaojing Wang Results 

Cut out KOSM from pLVX2MKOSiE 314 using Nde1 + BamH1, the insert is 6.5 kb. Subclone into Nde1 + BamH1 digested pAd5-CMV-MCS 280.

[[Image:314 280 digest 12.16.2011.JPG]]

ligation, transformation

select 5 clones to mini-prep

519 plasmids are digested by Nde1+BamH1,519-1,-3,-4 plasmids may be correct. 

[[Image:519digest f 1.4.2012 s.JPG]]

 

519-1,-3,-4 plasmids are digested by xho1. 

[[Image:519digest xho1 1.5.2012 s.JPG]]

[[Category:Cloning]]

Transfection reagents 2012-1-5

2012-01-09T23:14:28Z

Yingying.lu:

=== Guanidinylation ===

{| width="600" cellspacing="1" cellpadding="1" border="1"
|-
|
Gu D4 6K, undiluted 

| Gu D4 6K, 1:5
| Gu D4 6K, 1:10
| 528-1 
|-
| Gu D4 40K, undiluted
| Gu D4 40K, 1:5
| Gu D4 40K, 1:10
| 2011-11-10
|-
| PHP-ss-guPEI18, 1:40
| PHP-ss-guPEI18, 1:50
| guPEI18, 1:40
| guPEI18, 1:50
|}

24 hours after transfection

 

[[Image:2012 1 7 transfection reagents.jpg]] 

Day2

    [[Image:2012 1 8 transfection reagents.jpg]]

 

day 3

[[Image:Day 3 1.9.2012 s.JPG]]

[[Category:Transfection]]

File:Day 3 1.9.2012 s.JPG

2012-01-09T23:12:59Z

Yingying.lu: uploaded a new version of "File:Day 3 1.9.2012 s.JPG"