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Mutagenesis not working


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5 replies to this topic

#1 calix23

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Posted 28 October 2010 - 01:06 PM

I have tried doing a mutagenesis a few times but i haven't been able to get any colonies.

In the PCR I use:
10uL buffer
6uL (150ng) parent plasmid (~7k)
6uL (3uM final concentration) each primer (Tm = 66 from idtdna)
2uL Vent
6uL (300uM final concentration) dNTPs
70 water

Plasmid were made using dnassist

PCR Program:

cycle 1: 5 minutes @ 95
cycle 2: 50s @95, 50s @ 61, 8min @ 68, repeat 15 times
cycle 3: 7 minutes @ 68

After this I run a digestion by adding 2uL of DpnI into the reaction and leaving it overnight at 37

after the transformation i get no colonies. I use BL21 cells and pCWori as the vector.

I am very new to the biochemistry field so I don't know exactly what to do.
thanks for the replies

#2 phage434

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Posted 28 October 2010 - 07:15 PM

You should never try to transform BL21 cells following a ligation or mutagenesis reaction. Clone into a cloning strain such as Top10 or DH5a, then validate that your reaction has worked by sequencing miniprep'd DNA. Only then, transform into BL21 for expression. BL21 strains have very poor transformation efficiency.

#3 donny

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Posted 28 October 2010 - 07:51 PM

I would try using 50ng parent plasmid and 0.2uM final concentration of each primer.

#4 calix23

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Posted 29 October 2010 - 12:09 PM

I will get the results of the transformation later today (did transformation using DH5a cells 50ul and 10uL of PCR rxn i had in the freezer). Just in case, I am doing a PCR with less parent DNA and less primers as suggested.

Thanks for the replies

#5 calix23

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Posted 01 November 2010 - 06:08 AM

so the one I transformed into DH5a didn't work. I used 50uL of cells and 20uL of PCR rxn.

This is how I transform:

Incubate competent cells on ice for 5 min,
Add plasmid and pipet up and down four times,
Incubate on ice for 10 min,
heat shock for 20 sec @42,
incubate on ice for 2 min,
add rxn in 0.5mL nZY,
shake at 37 for 30-60min (i usually do 45),
pipet 150uL into plates with antibiotics.

plates i use are LB + ampicilin. As my plasmid has that resistance.

thanks.

#6 calix23

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Posted 01 November 2010 - 06:08 AM

so the one I transformed into DH5a didn't work. I used 50uL of cells and 20uL of PCR rxn.

This is how I transform:

Incubate competent cells on ice for 5 min,
Add plasmid and pipet up and down four times,
Incubate on ice for 10 min,
heat shock for 20 sec @42,
incubate on ice for 2 min,
add rxn in 0.5mL nZY,
shake at 37 for 30-60min (i usually do 45),
pipet 150uL into plates with antibiotics.

plates i use are LB + ampicilin. As my plasmid has that resistance.

Is this how I should transform DH5a?

thanks.

Edited by calix23, 01 November 2010 - 06:20 AM.





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