pCDF6 cloning with three inserts

on Sunday, November 8th, 2020 7:33 | by Sarah-Lynn Stratil

pCDF6 digestion with enzyme Bbs1
-5.5µl vector (undigested), conc.: 539.1 ng/µl
-1 µl enzyme (Bbs1)
-5 µl buffer (CutSmart)
-38.5 µl H2O
Agarose-gel electrophoresis
E.Z.N.A. Gel Extraction-DNA Purification from Agarose gel

7Concentration: 38,0 ng/µl
pCDF digested
concentration: 38,0 ng/µl

PCR pCDF6 Primer
-12.5 µl primer reverse/forward
-5 µl dNTPs
-2.5 µl Taq-Polymerase
-0.29 µl pCDF6 digested
-50 µl buffer
-167.5 µl H2O
Agarose-gel electrophoresis
E.Z.N.A. Gel Extraction

Concetration:
primer 1: 170.7 ng/µl
primer 2: 137.4 ng/µl
primer 3: 215.0 ng/µl
primer with 100kb ladder

pCDF6 NEBuilder Assembly Reaction
-pcr1: 4.85 ng -> 1.60 µl
-pcr2: 4.24 ng -> 1.20 µl
-pcr3: 4.87 ng -> 1.20 µl
-pCDF6: 100 ng -> 2.90 µl
-H2O: 3.1 µl
-10 µl NEBuilder HiFi DNA Assembly Master Mix/Control

heat shock transformation
-> plated on LB0+Amp plates
-> no colonies

colony PCR (29.10.20)
-37 µl primer forward/reverse
-37 µl dNTPs
-37 µl Taq Polymerase
-74 µl buffer LSB
-518 µl H2O
Agarose-gel electrophoresis


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Category: Foxp

One response to “pCDF6 cloning with three inserts”

  1. Ottavia Palazzo says:

    Is 100 bp, not 100 kb ;)

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