Team:Heidelberg/Templates/Del week15 pSB4K5

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Contents

07-08-2013

Results Sequencing

Send the backbone pSB4K5 from the partsregistry to GATC for sequencing:

  • pSB4K5 with Primer FS_01
  • pSB4K5 with Primer FS_16

Alignment of pSB4K5(FS_01 to FS_16) with Primer FS_01

Sequencing Result pSB4K5 FS 16 fw.clustal

.txt Alignment of pSB4K5(FS_01 to FS_16) with Primer FS_16

Results:

  • The sequence is the same as expected and indicated in the partsregistry.

Amplification from FS_01 to FS_16; 4.2 kb

Amplifcation of DelG (SR01-FS23), pSB4K5 and DelAFII; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelG (SR01-FS23), pSB4K5 and DelAFII cut; run at 100 V, 0.8 % gel (TAE)(07.08)
Reaction

(2x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad T100
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 ↓ 0.5 5
72 1:30 min
18 98 1
60 5
72 1:30 min
1 72 5 min
1 8 inf

Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

08-08-2013

Concentration measurement

The concentration of the gel purified fragment was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 FS16-FS01 07-08-2013 55.9 ng/µl
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