Have you ttake into account the size of your insert?

m also doing cloning experiments. Some times it happened bc of antibiotic selection and its conc as well so Badia Barakat rightly suggested.

Hope by doing this u will find solution of ur problem

Good Luck

Have you run a gel with your PCR products to make sure you have plasmid DNA in your samples? If the concentration is low you may have to add a larger volume of the PCR product to the transformation.

which cells and vector you are using for transformation. If you are transforming plasmid in expression host, maybe cells don't like your plasmid due to leaky expression. If u r doing with DH5a, go for e.coli top 10.

Hi, I was wondering what are you transfecting. if its a PCR product isolated from a gel I would go back and look into the ligation. sometimes the buffers involved in the purifying process of PCR products from a gel can inhibit a ligation. Also, the insert if PCR product sometimes does not have the correct ends for a ligation better to be able to cut the insert with a RE, and then use it for ligation. The fact that your controls do grow means that the problem is not your competent cells.

can you explain in detail what your goal is. Are you cloning PCRs in Puc18 ?

It may be useful if you give a step-by-step account of what you are doing so that it's easier to troubleshoot.

Here is my step-by-step account for attempting to synthesize two complimentary oligonucleotides...

Our goal is to essentially swop the enzyme dihydrofolate reductase from Haloferax volcanii with E. coli in order to study the salt stability of the protein.

Prepared control reaction with

- 5 uL 10X reaction buffer

- 2 uL (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/uL)

- 1.25 uL (125 ng) of oligonucleotide control primer #1

- 1.25 uL (125 ng) of oligonucleotide control primer #2

- 1 uL of dNTP mix

- 39.5 uL of double-distilled water to a final volume of 50 uL

Prepared the sample reaction with

- 5 uL of 10X reaction buffer

- 1 uL of dsDNA template

- 0.5 uL HVO1

- 0.5 uL HVO2

- 1 uL dNTP

- brought total volume to 50 uL with 42 uL double-distilled water

PCR Procedure

- segment 1

- One cycle with temp at 95 C for 30 seconds

- segment 2

- 16 cycles with temp at 95 C for 30 sec then 55 C for 1 min then 68 C for 1 minute

Plasmid of the selected DHFR Prep concentration is 75 ug/mL so I used 1 uL of Plasmid in the sample reaction

I used Dpn1 cells as our supercompetent cells to which I prepared them by

- pulled from freezer

- quick easy pulse spin

- 1 uL of Dpn1 into each microfuge tube

- incubated Dpn1 @ 37 C for 1 hour

- put on ice

- took 3 polypropelene tubes (2059), put on ice to chill

- warmed LB broth at 42 C

- quick gentle pulse spin of cells

- 65 uL of cells into three falcon tubes labeled

- pUC18

- Mutagenesis control

- Sample

- 1 uL of Beta Mercaptoethanol into each tube

- mix gently by swirl

- on ice for 10 minutes

- every 2 minutes swirl gently

- add 5 uL of control and mutagenesis sample to cells

- add 1 uL of pUC18 into tube labeled pUC18

- swirl gently

- back on ice for 30 minutes

- put tubes in water bath @ 42 C for 45 seconds then back on ice

- back on ice for 2 minutes

- add 0.5 mL LB broth with no antibiotics present

- incubate @ 37 C for 1 hour @ 225-250 rpm

- streak on plates

Plates streaked

- sample:

- 50 uL sample on LB broth and 1:1000 Ampicillin

- 200 uL sample on LB broth and 1:1000 Ampicillin

- Mutagenesis control:

- 50 uL mutagenesis control on LB, 1:1000 Amp, 0.4 M isopropylthio-β-galactoside, 80 ug/mL of X-gal

-200 uL mutagenesis control on LB, 1:1000 Amp, 0.4 M isopropylthio-β-galactoside, 80 ug/mL of X-gal

-pUC18:

- 50 uL mutagenesis control on LB, 1:1000 Amp, 0.4 M isopropylthio-β-galactoside, 80 ug/mL of X-gal

-200 uL mutagenesis control on LB, 1:1000 Amp, 0.4 M isopropylthio-β-galactoside, 80 ug/mL of X-gal

Let incubate for 16 hours at 37 C

What was observed is that there was no growth for the Sample AND Mutagenesis Control. The pUC18 grew with a 99.33% mutagenesis efficiency. I repreated this procedure after the PCR reaction and had the same results, this leads me to believe it may be due to a higher concentration of antibiotics than the supercompetent cells may handle (thanks to Badia Barakat). If anyone else has any other ideas I'd be happy to hear them! Thanks thus far, will be testing here shortly with a much lower antibiotic concentration to see if that will fix the issue.

Amanda Storm I had considered a low concentration so I repreated the process with 25 uL of the PCR product instead of the previous 5 uL and used 100 uL of cells into each falcon tube intead of the previous 65 uL. Sadly, same results followed.

Rina Yamin I will definitely look into that, thank you!

We are usually using Stratagene quick change mutagenesis kit. You should use XL1 blue competent cells

I appologize, I was trying to do that by memory (was away from the lab). You are correct Valerie, I used XL1 blue competent cells with the Stratagene QuikChange Mutagenesis Kit.

You could try adding an extra minute to the extention time. So instead of using 1minute per Kb use 1 minute per Kb plus an additional minute. This seems to be more of a necessity with larger rather than smaller constructs.

It seems to me, if I understand your conditions correctly, that the cells are fine since the puC18 worked. Is your mutagenesis construct particularly large? If this is the case, you might want to consider using XL10 gold cells, which are much better for tricky constructs.

Also, XL1-blue cells do not need to be treated with BME. It looks like you are using a protocol that is a hybrid between the Stratagene XL10 and XL1 protocols. Make sure you used the correct cells with the correct heat pulse times. The mutagenesis constructs might be more difficult to transform, and this may explain why one will work with an unusual protocol but the others won't.

If it turns out to be the mutagenesis (although this seems to not be the case either if the mutagenesis control isn't working), you can also try adding a little DMSO to increase the efficiency of you mutagenesis reaction. I've also found that adding a 20 min, 68 deg C hold at the end of your cycling helps efficiency.

Good luck! PS, if you really really need to get this done, you can send your construct to Mutagenex. They can do a single mutation for about $200. Turnaround time is ~ 2 weeks.