IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/12

From OpenWetWare

Jump to: navigation, search
UIUC - Arsenic Collection System! Main project page
Previous entry      Next entry

7/12/10

Gel

Lane Sample Amount
1 LamBF1R 5 ul sample, 1 ul dye
2 LamBF2R 5 ul sample, 1 ul dye
3 ArsR Fusion 5 ul sample, 1 ul dye
4 ArsR 5 ul sample, 1 ul dye
5 pArsRF1R1 5 ul sample, 1 ul dye
6 pArsRF1R2 5 ul sample, 1 ul dye
7 pArsRF2R1 5 ul sample, 1 ul dye
8 pArsRF2R2 5 ul sample, 1 ul dye
9 negative control 5 ul sample, 1 ul dye
10 1 Kb Ladder 1 ul

Faint bands not visible on picture for pArsRF2R1, pArsRF2R2, LamBF1R, LamB F2R Image:UIUC-Bioware7.12.10_(2pm)_20ng_PCR_lamB,_arsR,_pArsR.jpg


Lost pArsRF1R, pArsRF1R2 in PCR cleanup.

Eluted PCR cleanup into 2 different tubes at different volumes (50 and 20 μL):

pArsRF1R1(50ul) 5.4 ng/μL

pArsRF1R2 (50ul) 4.9 ng/μL

ArsR (50) 12 ng/μL

pArsRF1R1 (20ul) 1.8 ng/μL

pArsRF1R2 (20ul) 3.2 ng/ul

ArsR (20 ul) 4.8 ng/μL


Digestions

ArsR

DNA 41.67 ul (500ng)
H2O .83 ul
Buffer 2 5 ul
BSA .5 ul
EcoRI 1 ul
SpeI 1 ul

pSB1A2

DNA 3.93 ul (500 ng)
H2O 38.57 ul
Buffer 2 5 ul
BSA .5 ul
EcoRI 1 ul
SpeI 1 ul

Incubate at 37°C for 15 minutes. Inactivate at 80°C for 20 minutes.

Gel/Gel Purification:

Lane Sample Amount
1 1 kB ladder 1 ul
4 pSB1A2 entire sample plus dye
5 ArsR entire sample plus dye

NB: other lanes held Tom and Amanda's samples. Ran at 175V

Purification yields:

ArsR 56.8 ng/ul

pSB1A2 8 ng/ul

Ligation

1:6 ratio of vector:insert

H20 13 μL
pSB1A2 2 μL
ArsR 2 μL
10X Ligase Buffer 2 μL
Ligase 1 μL

Incubate 14°C overnight, inactivate (7/13/10 9:15AM) 80°C 20 min.


Personal tools