ELN - User contributions [en]

File:Plate3 2011 11 30.jpg

2011-11-30T15:31:42Z

Luyingying:

File:Plate2 2011 11 30.jpg

2011-11-30T15:31:33Z

Luyingying:

File:Plate1 2011 11 30.jpg

2011-11-30T15:31:23Z

Luyingying:

File:491 subclone 11-29-2011.JPG

2011-11-29T21:37:56Z

Luyingying:

File:475 clone 11-29-2011.JPG

2011-11-29T20:35:10Z

Luyingying:

474-pAd5.CMV-Cre-RFP

2011-11-29T19:06:38Z

Luyingying:

cloning of 474-pAd5-CMV-Cre-RFP

Purpose: to subclone from 469pAAV.CMV-Cre-RFP to 280pAd5-CMV-MCS 

Date: 2011-11-21----

Notebook:Lu yingying

Page:

Clone ID:474

Experimenters: lu yingying Results [[Image:474 subclone11-21-2011.JPG]] 

digest :EcoR1 + Xho1

band show insert and vector 

[[Image:367+474 digest detect11-23-2011.JPG]] 

digest detect :EcoR1 + Xho1 

[[Category:Cloning]]

File:475 PCR11-29-2011.JPG

2011-11-29T19:03:40Z

Luyingying:

File:457+461 clone 11-29-2011.JPG

2011-11-29T18:02:32Z

Luyingying:

456 pLex-CMV-CuO-Dest

2011-11-28T20:33:35Z

Luyingying:

Cloning of pLex-CMV-CuO-Dest (456)

Purpose: to clone pLex-CMV-CuO-Dest by PCR

Date: 2011-11-3 to 2011-11-

Notebook: Xiaojing Wang

Page: 004113---004114

Clone ID: 456

Experimenters: Xiaojing Wang

Results 

[[Image:456 PCR 11.3.2011.JPG|141x285px|456 PCR 11.3.2011.JPG]] 

The bands show PCR product of 456,

Primer:

GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG

GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG

Template is 392 pLVB-V5-Dest

Digest result:  

[[Image:456 PCR digest 11.3.2011.JPG|155x381px|456 PCR digest 11.3.2011.JPG]] 

digest PCR product and vector with Spe1 + Mlu1,run gel

gel purify, ligate, transform ccdb survival

 Colony Screening 

 

[[Image:456 clone digest 11.11.2011.JPG]]

Select 5 clones to mini-prep, digestd by Spl I and Mlu I.  456-1,-3.-4 may be correct. 

pDonr221_EGFP(393) + pLex-CMV-CuO-Dest(456-1/456-3/456-4)   make gateway by LR Clonase II. 

Transform 393+456-1/456-3/456-4 by STBL3, Amp. 

Select 4 clones each plate to mini-prep. The total number of plasmids is 12 (No.1-12).

 

[[Image:393+456 gateway by xiaojing 11.18.2011.jpg]]

No.1-12 plasmids transfect 293T cell line. 

 48 hours after transfection, take pictures.

 

[[Image:393+456 Digest 11.18.2011.JPG]]

No.1-12 plasmids are digested by BamHI. Bands 13 and 14 are control.

Band 13 is 456-3 digested by BamHI, and band 14 is 456-3.

No.1-5,7-9,11,12 may be correct.

 

Date: 2011-11-7 to 2011-11-10

Notebook: Yingying Lu 

Page: 

Clone ID: 456

Experimenters: Yingying Lu 

Results 

[[Image:456 pcr11-7-2011.JPG]] 

GW-SpeI-F agtac ACTAGT ATCAACAAGTTTGTACAAAAAAG

GW-Mlu1-R (CTTTCTTGTACAAAGTGGTTGAT CAC CAt CAC CAt CAC CAt tga ACGCGT atcta ) tagatACGCGTtcaaTGGTGaTGGTGaTGGTGATCAACCACTTTGTACAAGAAAG

Template is 392 pLVB-V5-Dest 

[[Image:456+493 clone11-8-2011.JPG]] 

digest PCR product and vector448pLex-CMV-CuO with Spe1 + Mlu1,run gel 

band 1 show insert  ,band 2 show vector 

[[Image:418+419+456 digest detect11-10-2011.JPG]] 

5 clonies digest detect:Spe1 + Mlu1, 456-3,456-5 may be right 

 

[[Image:393+456 gateway by yingying 11.18.2011.jpg]]

393EGFP to 456-5  5 clonies  transfect into cells 

[[Image:393+456 digest detect11-16-2011.JPG]] 

393-pDonr221_EGFP to 456-5 pLex-CMV-CuO-Dest 

then digest detect with BamH1 

Band 1---5 show 5 clones, band 6 show 456-5 pLex-CMV-CuO-Dest 

 

[[Category:Cloning]]

File:454 digest detect11-27-2011.JPG

2011-11-28T17:53:09Z

Luyingying:

File:493 insert+vector11-28-2011.JPG

2011-11-28T17:49:05Z

Luyingying:

Transfection reagents comparison 14

2011-11-28T14:54:58Z

Luyingying:

*'''Make 7 different transfection reagents (TR).'''

3 ul TR + 50ul DMEM without serum---->Mix A

1 ug DNA(pCMV-EGFP) + 50ul DMEM without serum---->Mix B

Mix A + Mix B, incubate 20min at room temperature

Add 100 ul mixture to transfect 293T cells in 12 well-plate. 

1-1,1-2,2-1,2-2,3-1 are EG-1031, lot 20111107.

4-1,4-2 are lot 20110930.

 

 

[[Image:SEVEN TR 11.4.2011.JPG|502x384px|SEVEN TR 11.4.2011.JPG]]

48 hours after transfection, picture taken on 11-4-2011 

 

[[Image:SEVEN TR 11.7.2011.JPG]] 

48 hours after transfection, picture was taken on 11-7-2011. 

 

 

*'''Make 4 different transfection reagents (TR). '''

3 ul TR + 50ul DMEM without serum---->Mix A

1 ug DNA(pCMV-EGFP) + 50ul DMEM without serum---->Mix B

Mix A + Mix B, incubate 20min at room temperature

Add 100 ul mixture to transfect 293T cells in 12 well-plate.

1 is EG-1086, lot 20110714.

2 is EG-1074, lot 20110803 (Gene Expresso 8000).

3 is EG-1031, lot 20111107.

4 is EG-1086T, lot 20111110. 

[[Image:11.11.2011.jpg]] 

 

48 hours after transfection, picture was taken on 11-11-2011. 

 

[[Category:Transfection]]

Transfection reagents freeze-thaw 100 times

2011-11-26T16:21:10Z

Luyingying:

Make transfection reagent (TR) to freeze-thaw 100 times.

3 ul TR + 50ul DMEM without serum---->Mix A

1 ug DNA(pCMV-EGFP) + 50ul DMEM without serum---->Mix B

Mix A + Mix B, incubate 20min at room temperature

Add 100 ul mixture to transfect 293T cells in 12 well-plate. 

0 is control.

 

 

48 hours after transfection 

[[Image:Plate3s 11.23.2011.jpg]] 

[[Image:Plate3s 11.23.2011.JPG]] 

2011-11-24 (3 days after transfection)

[[Image:2011 11 24 freeze thaw stability.jpg]] 

[[Image:2011_11_26_freeze_thaw_stability.jpg]]

--[[User:Luyingying|Xiaojing Wang]] 12:04, 23 November 2011 (EST) 

[[Category:Transfection]]

Transfection reagents 2011-11-21

2011-11-26T16:20:11Z

Luyingying:

Plate 1 

{| cellspacing="1" cellpadding="1" border="1" style="width: 552px; height: 159px;"
|-
| PHP-DSP-PEI 800, 1:10 
| PHP-DSP-PEI 800, 1:20 
| PHP-DSP-PEI 800, 1:30 
| PHP-DSP-PEI 800, 1:40 
|-
| PHP-DSP-PEI 1200, 1:10 
| PHP-DSP-PEI 1200, 1:20 
| PHP-DSP-PEI 1200, 1:30 
| PHP-DSP-PEI 1200, 1:40 
|-
| PHP-DSP-PEI 1800, 1:10 
| PHP-DSP-PEI 1800, 1:20 
| PHP-DSP-PEI 1800, 1:30 
| PHP-DSP-PEI 1800, 1:40 
|}

 

Plate 2 

{| cellspacing="1" cellpadding="1" border="1" style="width: 557px; height: 131px;"
|-
| PHP-DSP-PEI 800, 1:50 
| PHP-DSP-PEI 800, 1:60 
| PHP-DSP-PEI 800, 1:70 
| PHP-DSP-PEI 800, 1:80 
|-
| PHP-DSP-PEI 1200, 1:50 
| PHP-DSP-PEI 1200, 1:60 
| PHP-DSP-PEI 1200, 1:70 
| PHP-DSP-PEI 1200, 1:80 
|-
| PHP-DSP-PEI 1800, 1:50 
| PHP-DSP-PEI 1800, 1:60 
| PHP-DSP-PEI 1800, 1:70 
| PHP-DSP-PEI 1800, 1:80 
|}

 

 

48 hours after transfection 

Plate 1:

[[Image:Plate1 11.23.2011.jpg]]

[[Image:Plate1s 11.23.2011.JPG]]

 

Nov 24, 2011

[[Image:2011 11 24 compare 18 12 8 1.jpg]]

Nov 26, 2011 

[[Image:2011_11_26_compare_18_12_8_1.jpg]] 

Plate 2:

[[Image:Plate2s 11.23.2011.jpg]]

[[Image:Plate2s 11.23.2011.JPG]]

2011-11-24

[[Image:2011 11 24 compare 18 12 8 2.jpg]] 

[[Image:2011_11_26_compare_18_12_8_2.jpg]]

--Xiaojing Wang 11:39, 23 November 2011 (EST)

[[Category:Transfection]]

File:2011 11 26 compare 18 12 8 1.jpg

2011-11-26T16:16:55Z

Luyingying:

File:2011 11 26 compare 18 12 8 2.jpg

2011-11-26T16:16:45Z

Luyingying:

File:2011 11 26 freeze thaw stability.jpg

2011-11-26T16:15:24Z

Luyingying:

File:2011 11 24 freeze thaw stability.jpg

2011-11-24T15:28:09Z

Luyingying:

File:2011 11 24 compare 18 12 8 2.jpg

2011-11-24T15:27:55Z

Luyingying:

File:2011 11 24 compare 18 12 8 1.jpg

2011-11-24T15:27:42Z

Luyingying:

374-pAd5.CMV-Dest-His

2011-11-23T21:36:57Z

Luyingying:

Cloning of 374-pAd5.CMV-Dest-His

Purpose: to clone pAd5.CMV-Dest-His by gene synthesis 

Date: 2011-11-9----

Notebook:Lu yingying

Page:

Clone ID:374

Experimenters: lu yingying Results 

[[Image:374 PCR 11-9-2011.JPG]] 

PCR gateway cassette using 446-pVP16 vector as template 

ad-GW-F,ad-GW-His-R

[[Image:374 clone 11-9-2011.JPG]] cut with Kpn1 + Xho1 ligate to Kpn1 +Xho1 digested vector280 

 

Date: 2011-11-15----

Notebook:Lu yingying

Page:

Clone ID:374

Experimenters: lu yingying Results 

[[Image:374 PCR 11-15-2011.JPG]] 

PCR gateway cassette using 446-pVP16 vector as template

ad-GW-F,ad-GW-His-R 

[[Image:374 digest11-15-2011.JPG]] 

cut with Kpn1 + Xho1 ligate to Kpn1 +Xho1 digested vector280 

[[Image:374 digest detect11-17-2011.JPG]] 

5 different clones digest detect:Kpn1 + Xho1 

[[Image:393+374 test11-21-2011.JPG]]

digest :BamH1

24 hours after transfection

[[Image:237 374 s 11.23.2011.jpg]] 

[[Image:237 374 s 11.23.2011 blue.JPG]] 

[[Category:Cloning]]

File:237 374 s 11.23.2011.jpg

2011-11-23T21:36:07Z

Luyingying: uploaded a new version of "File:237 374 s 11.23.2011.jpg"

File:237 374 s 11.23.2011 blue.JPG

2011-11-23T21:34:58Z

Luyingying: uploaded a new version of "File:237 374 s 11.23.2011 blue.JPG"

File:Freeze-thaw 2 weeks blue s.jpg

2011-11-23T21:15:38Z

Luyingying:

File:Freeze-thaw 2 weeks blue.jpg

2011-11-23T21:15:04Z

Luyingying:

File:Freeze-thaw 2 weeks s.jpg

2011-11-23T21:13:59Z

Luyingying:

PAd5.CMV-Cre-IRES-EGFP

2011-11-23T21:06:04Z

Luyingying: moved PAd5.CMV-Cre-IRES-EGFP to 237-PAd5.CMV-Cre-IRES-EGFP

#REDIRECT [[237-PAd5.CMV-Cre-IRES-EGFP]]

File:493 clone insert+vector11-23-2011.JPG

2011-11-23T21:03:16Z

Luyingying:

237-pAd5-Cre-GFP

2011-11-23T20:09:01Z

Luyingying:

Cloning of Cre by subclone 241 to 242 

Purpose: to clone 237-pAd5-Cre-GFP 

Date: 2011-10-14

Notebook: luyingying

Page:

Clone ID:237

Experimenters: lu yingying

Result 

[[Image:237subclone10-13-2011.JPG]]

band shows vector

[[Image:237vector10-14-2011.JPG]]

bands show cre 

 

Restart 

Date: 2011-10-18 --- 

Notebook: luyingying

Page:

Clone ID:237

Experimenters: lu yingying

Result 

[[Image:237 crePCR product10-18-2011.JPG]] 

PCR product 

298 plasmid as template 

primer:Lenti-Cre-F+Lenti-Cre-R 

25 cycles

[[Image:237 vector+cre digest 10-19-2011.JPG]] 

digest resulte:EcoR1+ Xho1 NEB enzyme 37 for 3 hours 

242 plasmid  digest as vector 

 

Cloning of Cre by subclone 298 pTriEx-HTNC (Cre) to 242 pAd5-CMV-2-IRES-GFP 

Purpose: to clone 237-pAd5-Cre-GFP

Date: 2011-10-25

Notebook: luyingying

Page:

Clone ID:237

Experimenters: lu yingying

Result 

[[Image:470+359+237cloning 10-25-2011.JPG]] 

band 4,5 show subclone result:EcoR1+ Xho1 

band 4 show 242 pAd5-CMV-2-IRES-GFP digest result 

band 5 show 298 pTriEx-HTNC (Cre) digest result 

[[Image:237 digest detect10-27-2011.JPG]] 

band 1-6 show digest detect result:EcoR1+ Xho1 

 

Date: 2011-11-10 ---

Notebook: luyingying

Page:

Clone ID:237

Experimenters: lu yingying

Result 

[[Image:237 crePCR product10-18-2011.JPG]]

PCR amplify His-Tat-NLS-Cre(298) as temlate, using Cre-EcoR1-F B1  + Cre-BglII-R B2 

 

[[Image:237+469 digest11-9-2011.JPG]]

PCR product with EcoR1 + BglII, digest pAd5-CMV-IRES-GFP with BamH1 + EcoR1 

band 1 show insert, band 2 show pAd5-CMV-IRES-GFP(242) vector digest 

[[Image:237+469+493 digest detect 11-14-2011.JPG]]

 digest detect:EcoR1+ Not1 

 

Date: 2011-11-16 ---

Notebook: luyingying

Page:

Clone ID:237

Experimenters: lu yingying

Result 

[[Image:237-PCR 11-16-2011.JPG]] 

PCR amplify His-Tat-NLS-Cre(298) as temlate, using Cre-EcoR1-F2 B1 + Cre-BglII-R B2 

[[Image:237 insert+vector11-17-2011.JPG]] 

PCR product with EcoR1 + BglII, digest pAd5-CMV-IRES-GFP with BamH1 + EcoR1 

[[Image:237 digest detect11-21-2011.JPG]]

digest test:EcoR1+Not1

 

 

[[Category:Cloning]]

237-pAd5.CMV-Cre-IRES-EGFP

2011-11-23T20:08:07Z

Luyingying: moved 237-pAd5-Cre-GFP to 237-pAd5.CMV-Cre-IRES-EGFP

#REDIRECT [[PAd5.CMV-Cre-IRES-EGFP]]

File:493 PCR 11-23-2011.JPG

2011-11-23T18:57:36Z

Luyingying:

File:367+474 digest detect11-23-2011.JPG

2011-11-23T18:57:23Z

Luyingying:

File:Plate3s 11.23.2011.jpg

2011-11-23T17:03:15Z

Luyingying:

File:Plate3s 11.23.2011.JPG

2011-11-23T16:44:18Z

Luyingying:

File:Plate2s 11.23.2011.JPG

2011-11-23T16:34:38Z

Luyingying:

File:Plate2s 11.23.2011.jpg

2011-11-23T16:29:41Z

Luyingying:

File:Plate1s 11.23.2011.JPG

2011-11-23T16:19:06Z

Luyingying:

File:Plate1 11.23.2011.jpg

2011-11-23T16:13:13Z

Luyingying:

Transfection reagents 2011-11-12

2011-11-22T22:23:45Z

Luyingying:

== '''Plate 1, PHP-DSP-PEI18 vs PHP-DSP-PEI12''' ==

{| cellspacing="1" cellpadding="1" border="1" style="width: 580px; height: 122px;"
|-
| PHP-DSP-PEI18, 1:10 
| PHP-DSP-PEI18, 1:20 
| PHP-DSP-PEI18, 1:30 
| PHP-DSP-PEI18, 1:40 
|-
| PHP-DSP-PEI12, 1:10 
| PHP-DSP-PEI12, 1:10 
| PHP-DSP-PEI12, 1:10 
| PHP-DSP-PEI12, 1:10 
|-
| PHP-DSP-PEI18, 1:50 
| PHP-DSP-PEI18, 1:30, 11/10/2011 
| PHP-DSP-PEI12, 1:50 
| PHP-DSP-PEI18, 1:30, 11/10/2011 
|}

 

36 hours after transfection

[[Image:Plate1 11.15.2011.jpg|600px|Plate1 11.15.2011.jpg]]

[[Image:2011 11 15 Plate 1 s.jpg]]

 

[[Image:Plate1 2011 11 15.jpg]] --[[User:Yingying.lu|Yingying.lu]] 10:00, 17 November 2011 (EST) 4days

[[Image:2011 11 17 plate1.jpg|600px|2011 11 17 plate1.jpg]]

 

== Plate 2, PHP-DSP-PEI18 vs PHP-DSP-PEI12 ==

{| cellspacing="1" cellpadding="1" border="1" style="width: 580px; height: 122px;"
|-
| PHP-DSP-PEI18, 1:75 
| PHP-DSP-PEI18, 1:100 
| PHP-DSP-PEI18, 1:125 
| PHP-DSP-PEI18, 1:150 
|-
| PHP-DSP-PEI12, 1:75 
| PHP-DSP-PEI12, 1:100 
| PHP-DSP-PEI12, 1:125 
| PHP-DSP-PEI12, 1:150 
|-
| PHP-DSP-PEI18, 1:200 
| PHP-DSP-PEI18, 1:30, 11/10/2011 
| PHP-DSP-PEI12, 1:200 
| PHP-DSP-PEI18, 1:30, 11/10/2011 
|}

36 hours after transfection 

[[Image:Plate2 11.15.2011.jpg|600px|Plate2 11.15.2011.jpg]] 

[[Image:2011 11 15 plate 2 s.jpg|600px|2011 11 15 plate 2 s.jpg]]

[[Image:Plate2 2011 11 15 s.JPG]]

 

4 days

[[Image:2011 11 17 plate2.jpg]] --[[User:Yingying.lu|Yingying.lu]] 10:00, 17 November 2011 (EST) 

== Plate 3, EDA-CBA-PET18 vs PHP-DSP-PEI18 ==

 

{| cellspacing="1" cellpadding="1" border="1" style="width: 580px; height: 122px;"
|-
| EDA-CBA-PEI18, 1:10 
| EDA-CBA-PEI18, 1:20 
| EDA-CBA-PEI18, 1:30 
| EDA-CBA-PEI18, 1:40 
|-
| EDA-CBA-PEI18, 1:50 
| EDA-CBA-PEI18, 1:75 
| EDA-CBA-PEI18, 1:100 
| EDA-CBA-PEI18, 1:150 
|-
| PHP-DSP-PEI18, 1:50 
| PHP-DSP-PEI18, 1:75 
| PHP-DSP-PEI12, 1:75 
| PHP-DSP-PEI18, 1:30, 11/10/2011 
|}

36 hours after transfection 

[[Image:2011 11 15 plate 3 s.jpg]] 

 

[[Image:Plate3 11.15.2011.jpg|600px|Plate3 11.15.2011.jpg]] 

[[Image:Plate3 2011 11 15.jpg]]

4 days 

[[Image:2011 11 17 plate3.jpg]] --[[User:Yingying.lu|Yingying.lu]] 10:00, 17 November 2011 (EST)

== Plate 4, Compare to Commercial TR ==

36 hours after transfection

{| cellspacing="1" cellpadding="1" border="1" style="width: 580px; height: 122px;"
|-
| Max, 2011/11/10 
| Lipofectamine 2000 
| 2020 
| Jarkat 
|-
| TKO 
| LT1
| TurboFect 
| Attractene 
|-
| Max, 2011/11/10 
| 3 
| XtremeGene 9 
| XtremeGene HP 
|}

 

[[Image:2011 11 15 plate 4 s.jpg]] 

 

[[Image:Plate4 11.15.2011.jpg|600px|Plate4 11.15.2011.jpg]]

[[Image:Plate4 2011 11 15.jpg]]

4 days

[[Image:2011 11 17 plate4.jpg]] --[[User:Yingying.lu|Yingying.lu]] 10:00, 17 November 2011 (EST)

[[Category:Transfection]]

Transfection reagents comparison 16

2011-11-22T20:38:35Z

Luyingying:

[[Image:Protein one transfection comparison.jpg]]

 

[[Image:Compare 11 4 2011.jpg]]

[[Image:Protein One.jpg]] 

 --[[User:Luyingying|Luyingying]] 17:00, 4 November 2011 (EST)

Transfection reagents comparison Proteinone

2011-11-22T20:38:23Z

Luyingying: Created page with "Image:Protein one transfection comparison.jpg Image:Compare 11 4 2011.jpg Image:Protein One.jpg Category:Transfection"

[[Image:Protein one transfection comparison.jpg]]

[[Image:Compare 11 4 2011.jpg]]

[[Image:Protein One.jpg]]

[[Category:Transfection]]

Transfection reagents comparison 15

2011-11-22T20:32:43Z

Luyingying:

Make transfection reagent (TR) to freeze-thaw ten times.

3 ul TR + 50ul DMEM without serum---->Mix A

1 ug DNA(pCMV-EGFP) + 50ul DMEM without serum---->Mix B

Mix A + Mix B, incubate 20min at room temperature

Add 100 ul mixture to transfect 293T cells in 12 well-plate.

 

[[Image:TR FREEZE 0-10 11.4.2011.JPG|629x481px|TR FREEZE 0-10 11.4.2011.JPG]] 

48 hours after transfection, picture taken on 11-4-2011 

[[Image:TR FREEZE 0-10 11.7.2011.JPG]] 

Picture was taken on 11-7-2011.

Transfection reagents freeze-thaw 10 times

2011-11-22T20:31:47Z

Luyingying: Created page with "Make transfection reagent (TR) to freeze-thaw 10 times. 3 ul TR + 50ul DMEM without serum---->Mix A 1 ug DNA(pCMV-EGFP) + 50ul DMEM without serum---->Mix B Mix A + Mix B..."

Make transfection reagent (TR) to freeze-thaw 10 times.

3 ul TR + 50ul DMEM without serum---->Mix A

1 ug DNA(pCMV-EGFP) + 50ul DMEM without serum---->Mix B

Mix A + Mix B, incubate 20min at room temperature

Add 100 ul mixture to transfect 293T cells in 12 well-plate.

[[Image:TR FREEZE 0-10 11.4.2011.JPG]]

48 hours after transfection, picture taken on 11-4-2011

[[Image:TR FREEZE 0-10 11.7.2011.JPG]]

Picture was taken on 11-7-2011.

 

[[Category:Transfection]]

File:Protein One.jpg

2011-11-22T20:09:49Z

Luyingying:

File:454 clone 11-22-2011.JPG

2011-11-22T16:45:36Z

Luyingying:

File:11.11.2011.jpg

2011-11-22T16:34:10Z

Luyingying: uploaded a new version of "File:11.11.2011.jpg"

File:454 digest detect11-21-2011.JPG

2011-11-21T21:25:10Z

Luyingying:

File:237 digest detect11-21-2011.JPG

2011-11-21T21:24:57Z

Luyingying:

File:393+374 test11-21-2011.JPG

2011-11-21T21:24:42Z

Luyingying:

File:367PCR 11-18-2011.JPG

2011-11-21T18:58:14Z

Luyingying: