Team:Heidelberg/Templates/Del week15 FG

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Contents

08-08-2013

Restriction digest of fragment from FS_21 to FS_26; 5.5 kb; 29-07-2013) with ClaI

Restriction digest of DelG (SR01-FS23), DelG (FS21-FS26) and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)

Incubation at Room temperature for about 4h and at 37°C for 1 hours

what µl
FS_21 to FS_26 (29-07-2013) 10
ClaI 1
Buffer CutSmart 1.5
dd H2O 2.5
Expected fragment lengths [bp] 2743, 1519, 1208

Results:

  • restriction digest of DelFG led to the expected fragment sizes, therefore restriction digest, implies amplification of the correct fragment
  • PCR product will be further validated by single read sequencing before Gibson Assembly

Concentration measurement DelFG

Fragment Primer Date PCR Concentration
DelFG FS21-FS26 07-08-2013 24.3 ng/µl

09-08-2013

Amplification from FS_21 to FS_26; 5.5 kb

Amplifcation of DelOP (I), DelFG and DelL ; run at 100 V, 0.8 % gel (TAE)(09.08)
Amplifcation of DelOP (I), DelFG and DelL ; run at 100 V, 0.8 % gel (TAE)(09.08)
Reaction
what µl
D. acidovorans SPH-1 (colony-pcr) 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
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