If have difficulties in testing positive clones via colony PCR.
Context: I did a LR Gateway reaction between an Entry Vector and three different Destination vectors.
Result should be 3 different Expression vectors with the same gene insert each.
I did this with 2 gene inserts which should have the same sequences.
let's call them
Then I transformed these vectors into E.coli and grow them on selection plates. Because of the antibiotic resistance plus
the ccdB gene on the destination vector there is a double selection for positive clones there should be a high chance getting
After 1 night incubation on 37°C I picked single colonies and did a colony PCR to verify positive clones.
Therefore I first tested
with 3 colonies for each vector (= 9 PCR reactions)
8 out of nine tested positive.
Meanwhile I got the results from entry clone sequencing: Base exchanges in the gene insert.
So I transformed my backup (sequence of the entry clone before LR reaction seems to be correct):
I ran the same colony PCR procedure:
NO positive clone.
I repeated PCR, picked new clones, repeated LR reaction and transformation, but I never got positive
results of the colony PCR anymore...
Maybe there is a mistake in my procedure???
a) get single colonies
1) Pick single colony from plate with a pipette tip
2) scratch pipette tip in a 1,5 ml reaction tube containing 10 µl water
3) mix by pipetting
b ) prepare master mix
32.5 µl dH2O
5.0 µl Buffer
1.0 µl dNTPs 10 mM
5.0 µl forward Primer 5 µM
5.0 µl reverse Primer 5 µM
0.5 µl Taq Polymerase
∑ 49 µl
c) Put mastermix in PCR tubes and add 1 µl of the bacteria suspension to each tube
d) run PCR
1) 3 min 94 °C
2) 30 sec 94 °C
3) 30 sec 47 °C
4) 1:45 min 72 °C
5) 6 min 72 °C
6) ∞ 4 °C
Steps 2-4 are repeated 35 times
Length of the section to be amplified = 1403 bp
Maybe someone can support me with some ideas and improvements...
Edited by Ikar, 23 April 2011 - 11:36 AM.