IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-20: Difference between revisions
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==double digesting E0040 and I7100 for ligation== | ==double digesting E0040 and I7100 for ligation== | ||
*Digesting E0040 and I7100 with EcoR1/SpeI. | *Digesting E0040 and I7100 with EcoR1/SpeI. | ||
*Digestion system contains (totally | *Digestion system contains (totally 19uL): 2uL 10*H, 0.5uL EcoRI, 0.5uL PstI, 6uL plasmid, 10uL ddH2O. | ||
*37℃ culutre for 12 hours. | *37℃ culutre for 12 hours. | ||
===electrophoresis result=== | ===electrophoresis result=== | ||
| Line 15: | Line 15: | ||
===electrophoresis test the products of gel extraction=== | ===electrophoresis test the products of gel extraction=== | ||
*Digestion system contains:2uL plasmid,2uL Loading Buffer,2uL Dye. | |||
* from left to right: | * from left to right: | ||
#EGFP(E0040) | |||
EGFP(E0040) | #pSB3k3(I7100) | ||
#Marker DL200 plus | |||
pSB3k3(I7100) | [[Image:2007-7-20_EGFP(E0040)_and_pSB3k3(I7100).jpg]] | ||
Marker DL200 plus | |||
==ligation== | ==ligation== | ||
*ligate the pSB3k3(I7100) and EGFP(E0040) with T4 ligase | *ligate the pSB3k3(I7100) and EGFP(E0040) with T4 ligase | ||
==mini-prep I7100 and E0040== | ==mini-prep I7100 and E0040== | ||
==single digestion test | ==single digestion test== | ||
* | *Digesting E0040 and r0040 with EcoR1. | ||
* for | *Digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O. | ||
*37℃ culutre for 3 hours. | |||
===electrophoresis result== | |||
* from left to right: | * from left to right: | ||
#1-3 e0040 and EcoR1<br> | |||
1-3 e0040 and EcoR1 | #4-5 r0040 and EcoR1<br> | ||
#6 Marker<br> | |||
4-5 r0040 and EcoR1 | [[Image:E0040-3-4-5_EcoR1 and R0040-2-4_EcoR1.jpg]] | ||
6 Marker | |||
*! there are abnormal bands in the e0040 samples. | *! there are abnormal bands in the e0040 samples. | ||
==another single digestion test== | ==another single digestion test== | ||
Revision as of 06:56, 30 July 2007
double digesting E0040 and I7100 for ligation
- Digesting E0040 and I7100 with EcoR1/SpeI.
- Digestion system contains (totally 19uL): 2uL 10*H, 0.5uL EcoRI, 0.5uL PstI, 6uL plasmid, 10uL ddH2O.
- 37℃ culutre for 12 hours.
electrophoresis result
- from left to right:
- 1 I7100 plasmid
- 2-5 I7100 and EcoR1 and Pst1
- 6 E0040 plasmid
- 7-10 E0040 and EcoR1 and Pst1
- 11 marker DL200 plus
- 1 I7100 plasmid
Gel Purification
- extract pSB3k3(I7100) vector and e0040 fragments from gel
electrophoresis test the products of gel extraction
- Digestion system contains:2uL plasmid,2uL Loading Buffer,2uL Dye.
- from left to right:
- EGFP(E0040)
- pSB3k3(I7100)
- Marker DL200 plus
_and_pSB3k3(I7100).jpg)
ligation
- ligate the pSB3k3(I7100) and EGFP(E0040) with T4 ligase
mini-prep I7100 and E0040
single digestion test
- Digesting E0040 and r0040 with EcoR1.
- Digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O.
- 37℃ culutre for 3 hours.
=electrophoresis result
- from left to right:
- 1-3 e0040 and EcoR1
- 4-5 r0040 and EcoR1
- 6 Marker
- ! there are abnormal bands in the e0040 samples.
another single digestion test
- digest I7100 with Xba1
- digest e0040(another sample) with EcoR1
- electrophoresis results. From left to right:
1 I7100 plasmid
2-4 I7100 and Xba1
5 e0040 plasmid
6-8 e0040 and EcoR1
9 marker DL2000 plus
10 r0040 and EcoR1 (products of former digestion test)
