IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-26: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(New page: ==Ligation:crRNA->R0040== *Ligate the crRNA fragment and R0040 vector. *Ligation system contains :1uL crRNA, 7uL R0040, 1uL 10XT4L Buffer, 1uL T4 Ligase. * 4℃ culutre for 12 hours)
 
No edit summary
Line 3: Line 3:
*Ligation system contains :1uL crRNA, 7uL R0040, 1uL 10XT4L Buffer, 1uL T4 Ligase.
*Ligation system contains :1uL crRNA, 7uL R0040, 1uL 10XT4L Buffer, 1uL T4 Ligase.
* 4℃ culutre for 12 hours
* 4℃ culutre for 12 hours
=Lock & Key By Yu Tao=
==Double Digesting J23078==
*Digesting J23078 PCR product with EcoR1/PstI.
*Digestion system contains:
<pre>
2 µl      10*M buffer
0.5 µl    XbaI
0.5 µl    PstI
10 µl      PCR product
7 µl      ddH20
--------------------------
20 µl      Total
</pre>
*37℃ culture for 9 hours and 16℃ culture for 2 hours.
==J23078 and R0040 Digestion Product purification==
*use Transgen EasyPure PCR Purification Kit for J23078 and Quick Gel Extraction Kit for R0040.
*30uL after purflication, respectively.
===electrophorsis result===
*from ''left'' to ''right'':
#J23078 @ XbaI/PstI without Genefinder(DNA dye) but loading buffer newly prepared by ourselves added
#J23078 @ XbaI/PstI without Genefinder(DNA dye) but loading buffer newly prepared by ourselves added
#J23078 @ XbaI/PstI with Genefinder(DNA dye) and loading buffer newly prepared by ourselves added
#J23078 @ XbaI/PstI with Genefinder(DNA dye) and loading buffer newly prepared by ourselves added
#J23078 @ XbaI/PstI without Genefinder(DNA dye) but commercial loading buffer prepared by ourselves added
#J23078 @ XbaI/PstI without Genefinder(DNA dye) but commercial loading buffer prepared by ourselves added
#Precise Quantified MarkerI
#R0040 @ PstI/SpeI
[[Image:Peking_2007-7-26-J23078_PstI/XbaI.jpg‎]]
*Results are expected.
*I am determined to use the commercial loading buffer and to add DNA dye to the samples everytime from now.
==Ligation: J23078(crRNA) and R0040==
*Ligate the J23078 fragment and R0040 vector
*Ligation system contains:
<pre>
1 µl      J23078 fragment
7 µl      R0040 vector
1 µl      T4-Ligase
1 µl      10 X ligation buffer
0 µl      ddH20
--------------------------
10 µl      Total
</pre>
*4℃ overnight(11 hours), then to be at 16℃ for 6 hours.
==Transformation: R0010<-J23066==
*Transform 10 µl R0010<-J23066 ligation product into 100 µl DH5αcompetent cells.
*Culture all the cells at Amp+ LB plate for 12 hours.
*Results to be seen tommorow.
===Mini-prep preparation: R0010 and J23066===
*Culture R0010 and J23066 positive colonies in liquid LB overnight for mini-prep.

Revision as of 04:25, 9 August 2007

Ligation:crRNA->R0040

  • Ligate the crRNA fragment and R0040 vector.
  • Ligation system contains :1uL crRNA, 7uL R0040, 1uL 10XT4L Buffer, 1uL T4 Ligase.
  • 4℃ culutre for 12 hours

Lock & Key By Yu Tao

Double Digesting J23078

  • Digesting J23078 PCR product with EcoR1/PstI.
  • Digestion system contains:
2 µl       10*M buffer
0.5 µl     XbaI
0.5 µl     PstI
10 µl      PCR product
7 µl       ddH20
--------------------------
20 µl      Total
  • 37℃ culture for 9 hours and 16℃ culture for 2 hours.

J23078 and R0040 Digestion Product purification

  • use Transgen EasyPure PCR Purification Kit for J23078 and Quick Gel Extraction Kit for R0040.
  • 30uL after purflication, respectively.

electrophorsis result

  • from left to right:
  1. J23078 @ XbaI/PstI without Genefinder(DNA dye) but loading buffer newly prepared by ourselves added
  2. J23078 @ XbaI/PstI without Genefinder(DNA dye) but loading buffer newly prepared by ourselves added
  3. J23078 @ XbaI/PstI with Genefinder(DNA dye) and loading buffer newly prepared by ourselves added
  4. J23078 @ XbaI/PstI with Genefinder(DNA dye) and loading buffer newly prepared by ourselves added
  5. J23078 @ XbaI/PstI without Genefinder(DNA dye) but commercial loading buffer prepared by ourselves added
  6. J23078 @ XbaI/PstI without Genefinder(DNA dye) but commercial loading buffer prepared by ourselves added
  7. Precise Quantified MarkerI
  8. R0040 @ PstI/SpeI

File:Peking 2007-7-26-J23078 PstI/XbaI.jpg

  • Results are expected.
  • I am determined to use the commercial loading buffer and to add DNA dye to the samples everytime from now.

Ligation: J23078(crRNA) and R0040

  • Ligate the J23078 fragment and R0040 vector
  • Ligation system contains:
1 µl       J23078 fragment
7 µl       R0040 vector
1 µl       T4-Ligase
1 µl       10 X ligation buffer
0 µl       ddH20
--------------------------
10 µl      Total
  • 4℃ overnight(11 hours), then to be at 16℃ for 6 hours.

Transformation: R0010<-J23066

  • Transform 10 µl R0010<-J23066 ligation product into 100 µl DH5αcompetent cells.
  • Culture all the cells at Amp+ LB plate for 12 hours.
  • Results to be seen tommorow.

Mini-prep preparation: R0010 and J23066

  • Culture R0010 and J23066 positive colonies in liquid LB overnight for mini-prep.
Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-26&oldid=139035"