IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-24: Difference between revisions
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i will be back @29/July ,add oil ,all! | i will be back @29/July ,add oil ,all! | ||
==Purification of the Digested Fragment and Vector== | |||
*Fragment: crRNA(J23078); Vector: R0040. | *Fragment: crRNA(J23078); Vector: R0040. | ||
*Use PCR Purification Kit. | *Use PCR Purification Kit. | ||
*The volume of purified fragment and vector: each 30uL, respectively. | *The volume of purified fragment and vector: each 30uL, respectively. | ||
==Electorphoresis== | |||
*To check the yield of the purified fragment and vector. | *To check the yield of the purified fragment and vector. | ||
*2% agarose.(For the ~70bp crRNA) | *2% agarose.(For the ~70bp crRNA) | ||
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[[Image:crRNA and R0040 test.jpg]] | [[Image:crRNA and R0040 test.jpg]] | ||
==Ligation== | |||
*Ligate the crRNA fragment and R0040 vector. | *Ligate the crRNA fragment and R0040 vector. | ||
*crRNA 2uL, R0040 4uL, totally 10uL system. | *crRNA 2uL, R0040 4uL, totally 10uL system. | ||
*system without crRNA added as a negative control. | *system without crRNA added as a negative control. | ||
*4 centigrade degree, overday. | *4 centigrade degree, overday. | ||
*Then transform them. | |||
Revision as of 11:10, 24 July 2007
pls see my suggestion @notebook 2007-7-23 -by QuMingZhi i will be back @29/July ,add oil ,all!
Purification of the Digested Fragment and Vector
- Fragment: crRNA(J23078); Vector: R0040.
- Use PCR Purification Kit.
- The volume of purified fragment and vector: each 30uL, respectively.
Electorphoresis
- To check the yield of the purified fragment and vector.
- 2% agarose.(For the ~70bp crRNA)
- Each sample: 2uL; with 1uL 10*loading buffer and 1uL 200* DNA dye added, respectively.
- From left to right: crRNA_PstI+XbaI, R0040_PstI+SpeI, R0040 plasmid, Marker I.
Ligation
- Ligate the crRNA fragment and R0040 vector.
- crRNA 2uL, R0040 4uL, totally 10uL system.
- system without crRNA added as a negative control.
- 4 centigrade degree, overday.
- Then transform them.
