IGEM:Hong Kong UST 2014/2009/Notebook/ATP Concentration Effect to Ligation Speed/2014/06/04

From OpenWetWare

Jump to: navigation, search
ATP Concentration Effect to Ligation Speed Main project page
Previous entry      Next entry

Wednesday, 4th June 2014



3. Take out the inoculated RFP-pSB1C3 (1,2,3,4) at 9:00am 4. Centrifuge the RFP PSB1C3 (1,2,3,4) for 5 minutes, using 3500 rpm speed.

5. MiniPrep :
Plasmids were extracted using Gene Tech GTpure Plasmid MiniPrep Purification Kit. DNA then eluted in 50 μL DE Buffer (2 times)

6. NanoDrop : Use DE Buffer as Blank

Concentration
A260/A280
A260/A230
RFP PSB1C3 (1)
228.1
1.93
2.12
RFP PSB1C3 (2)
124.8
1.91
1.91
RFP PSB1C3 (3)
220.8
1.90
1.65
RFP PSB1C3 (4)
219.2
1.94
2.26


7. Digestion : EcoRI-HF & PstI-HF
Use Cutsmart buffer
Digestion Composition :
- 1 ug DNA (1000 ng)
- 2 uL of Cutsmart buffer
- 0.5 uL of EcoRI-HF
- 0.5 uL of PstI-HF
- ddH2O = Adjust volume to 20 uL
Incubate for 1 hour, 37 degree Celcius.

8. Gel Electrophoresis :
0.8% Agarose Gel, 20 ml, 6 wells

WELL 1 (LADDER):
- 1 uL Loading Dye
- 1 uL GenRuler 1 kb Ladder
- 8 uL ddH2O

WELL 2 (NEGATIVE CONTROL): - 1 uL of Uncut Plasmid RFP-pSB1C3(2) (124.8 ng/lane)
- 1 uL Loading Dye
- 8 uL ddH20

WELL 3-6 (DIGESTION PRODUCT)(1ug/lane):
- 20 uL of DNA
- 2.2 uL of 10X Loading Dye

Gel Photo


Ladder - Uncut RFP-PSB1C3 - EcoRI-HF and PstI-HF digested RFP-PSB1C3 (1-4)


9. Gel Extraction & Purification
Insert and backbone were extracted out from the gel using Favorgen FavorPrep GEL/PCR Purification Kit. The DNA is then eluted in 50 μL of Elution Buffer

10. NanoDrop



Concentration (nanogram/microliter)
A260/A280
A260/A230
RFP Insert 1
5.0
3.37
0.17
RFP Insert 2
4.0
2.74
0.07
RFP Insert 3
3.4
4.19
0.12
RFP Insert 4
7.0
2.28
0.04
PSB1C3 Backbone 1
7.1
2.51
0.21
PSB1C3 Backbone 2
5.9
3.09
0.13
PSB1C3 Backbone 3
3.3
10.81
0.40
PSB1C3 Backbone 4
5.8
3.63
0.06



Personal tools