Team:DTU-Denmark/Notebook/12 June 2013

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{{:Team:DTU-Denmark/Templates/StartPage|12 June 2013}}
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=208=
=208=
 +
==Main purposes today==
 +
New PCR amplification of USER fragments
 +
 +
==Procedure==
 +
===PCR===
 +
MasterMix x 17 was made
 +
to each tube 3ul of Fw and 3 ul of rv primer was added as well as 1ul DNA template
 +
 +
every fragment was run 2 min 98 C followed by 35 cycles of
 +
 +
10 sec 98 C
 +
 +
1 sec 65 C ramp by 0.1C/s down to
 +
 +
10 sec of 55C
 +
 +
3 min 72C
 +
 +
after repetitions, 5 min of 72 C
 +
===prepared===
 +
*1l of 10x TBE
 +
*200ml of 2% agarose solution and added 10 ul EtBr
 +
* casting 3x 2% gels and 1x1% gel
 +
* 200ml EDTA stock solution 0.5 M from powder, dissolved at pH 8 and later equilibrated to pH 7
 +
* 200ml LB medium w. agar and kanamycin
 +
*autoclaved 1.5l of LB medium
 +
*new batch of competent cells
 +
* glycerol stock from I14032
 +
 +
===run gel of todays PCR===
 +
got SFGFP for TAT and SFGFP for Sec

Revision as of 12:19, 17 July 2013

12 June 2013

Contents

208

Main purposes today

New PCR amplification of USER fragments

Procedure

PCR

MasterMix x 17 was made to each tube 3ul of Fw and 3 ul of rv primer was added as well as 1ul DNA template

every fragment was run 2 min 98 C followed by 35 cycles of

10 sec 98 C

1 sec 65 C ramp by 0.1C/s down to

10 sec of 55C

3 min 72C

after repetitions, 5 min of 72 C

prepared

  • 1l of 10x TBE
  • 200ml of 2% agarose solution and added 10 ul EtBr
  • casting 3x 2% gels and 1x1% gel
  • 200ml EDTA stock solution 0.5 M from powder, dissolved at pH 8 and later equilibrated to pH 7
  • 200ml LB medium w. agar and kanamycin
  • autoclaved 1.5l of LB medium
  • new batch of competent cells
  • glycerol stock from I14032

run gel of todays PCR

got SFGFP for TAT and SFGFP for Sec


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