Team:DTU-Denmark/Notebook/12 June 2013

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=208=
=208=
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<hr/>
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==Main purposes today==
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<hr/>
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New PCR amplification of USER fragments
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==Procedure==
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<hr/>
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===PCR===
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MasterMix x 17 was made
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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
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after repetitions, 5 min of 72 C
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===prepared===
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*1l of 10x TBE
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*200ml of 2% agarose solution and added 10 ul EtBr
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* casting 3x 2% gels and 1x1% gel
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* 200ml EDTA stock solution 0.5 M from powder, dissolved at pH 8 and later equilibrated to pH 7
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* 200ml LB medium w. agar and kanamycin
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*autoclaved 1.5l of LB medium
 +
*new batch of competent cells
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* glycerol stock from I14032
 +
 +
===run gel of todays PCR===
 +
got SFGFP for TAT and SFGFP for Sec
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Latest revision as of 21:00, 16 September 2013

12 June 2013

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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

Navigate to the Previous or the Next Entry

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