IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-20: Difference between revisions
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*Digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O. | *Digestion system contains (totally 20uL): 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O. | ||
*37℃ culutre for 3 hours. | *37℃ culutre for 3 hours. | ||
===electrophoresis result== | ===electrophoresis result=== | ||
* from left to right: | * from left to right: | ||
#1-3 e0040 and EcoR1<br> | #1-3 e0040 and EcoR1<br> | ||
| Line 38: | Line 38: | ||
*! there are abnormal bands in the e0040 samples. | *! there are abnormal bands in the e0040 samples. | ||
==another single digestion test== | ==another single digestion test== | ||
* digest I7100 with Xba1 | * digest I7100 with Xba1 | ||
* digest e0040(another sample) with EcoR1 | * digest e0040(another sample) with EcoR1 | ||
* electrophoresis | * Digestion system contains (totally 20uL): | ||
#I7100: 2uL 10*M, 0.5uL XbaI, 4uL plasmid, 13.5uL dH2O. | |||
1 I7100 plasmid | #E0040: 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O. | ||
===electrophoresis result=== | |||
2-4 I7100 and Xba1 | *From'' left'' to ''right'': | ||
#1 I7100 plasmid | |||
5 e0040 plasmid | #2-4 I7100 and Xba1 | ||
#5 e0040 plasmid | |||
6-8 e0040 and EcoR1 | #6-8 e0040 and EcoR1 | ||
#9 marker DL2000 plus | |||
9 marker DL2000 plus | #10 r0040 and EcoR1 (products of former digestion test) | ||
10 r0040 and EcoR1 (products of former digestion test) | |||
[[Image:I7100-3-4-5 XbaI and E0040-6-7-8 EcoRI and R0040-4 EcoRI.jpg]] | [[Image:I7100-3-4-5 XbaI and E0040-6-7-8 EcoRI and R0040-4 EcoRI.jpg]] | ||
Latest revision as of 09:20, 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
- Digestion system contains (totally 20uL):
- I7100: 2uL 10*M, 0.5uL XbaI, 4uL plasmid, 13.5uL dH2O.
- E0040: 2uL 10*H, 0.5uL EcoRI, 4uL plasmid, 13.5uL dH2O.
electrophoresis result
- 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)
