Are you using the same LB plates and antibiotics as your lab mates? How have you stored your competent cells (temp, aliquot size)? Have you checked to make sure the antibiotic resistance marker on the plasmid matches that used on the plates?

Grace,

I had experienced to transform pGEX and pUC19 into DH5-alpha each, ended up with no colony at 16, 20, 48 hours of incubation on LB contained appropriate concentration of AMP.

What I learned from previous experiment was that SOB+MgCl2+MgSO4+AMP medium enhanced significantly transformation efficiency, in particular for pUC19, rather than LB+AMP did. You could perhaps try the former medium to thrive transformed cell. Please find the detail recipes on Sambrook and Russell's Molecular Cloning (2001).

In handling sample, was there any difference in transformation techniques (e.g. pippetting) between the labmates and your self? If so then, that should be main consideration for you.

Best.

Thanks for all of your responses.

I have stored the CC at -80. I am using 2YT plates while my labmates prefer LB medium. In terms of antibiotic, they used my CCs on plasmid containing either amp or kanamycin resistance gene. In both cases, they were able to observe transformants. 

The pET vector that I'm using has an amp resistance gene and am using that on the plates as well (100 mg/ml). 

I am using SOC medium for the transformation. Regarding the technique, we have been doing the same thing since I was the one who gave them the protocol for the transformation using TSS competent cells.

May I ask for how long you are thawing your CC and also for how long you are giving them heat shock for transformation? Even I feel the way you do spread spreading also plays a role, avoid  too much drying of your plates while spreading plates, once you start feeling friction while plating the cells stop at that point and keep the plates inside incubator. Hope it helps. I have one question how did you confirm your ligation by PCR? even if insert is not ligated it will get amplifed  by PCR. are you sure ligation is successful ?

Grace

I was struggling with the same problem lately,but after I modified my transformation protocol I got good number of colonies.you can try after heat shock for 50 sec at 42 degress incubate on ice for 2 min then add 400 microlitres of Lb without ampicillin and incubate at 37 degress shaking and den centrifuge at 3000 g for 5 min and remove 400 microlitres of supernatant and resuspend pellet in remaining left over supernatant and then spread whole on LB agar plates with specific antibiotic .. Hope this is of help...as it helped me. 

Do check antibiotic resistance of your plasmid and use the same antibiotic. 

Amp concentration should be 100 micrograms per mL in the plates, not 100 milligram per mL. If this was merely a typo, OK, but if not, it accounts for your problem. Home-made competent cellsd can have widely varying CFUs, depending on the strain and the way they were prepared. Anywhere from 10^5 to 10^7 CFU per ug can be fairly expected. For your positive control pUC19 transformation, I would keep the total amount of plasmid to 1-10ng per transformation. Make sure you plate sufficient volumes to see colonies even for the low end of transformation efficiency.

If you can't get anywhere with the pUC19 positive control, you should ask one of your labmates to do the procedure in parallel with you to try and see if there is a technical difference in the approach that accounts for the differences in success rate. Also see if you can get another batch of competent cells to help you troubleshoot.

To get better help on this forum, please post the bacterial strain you are using as well as the full details of your transformation protocol.

Good luck!

Thanks again for all the answers.

Regarding the time of thawing, I usually thaw my CCs on ice around 20-30 min then flick the tube a few times to mix them before getting 100 microliter for one transformation reaction. I did the PCR on the plasmid using the sequencing primers included in the pET kit, which was the T7 primer pair. When I used them on my ligation reaction, I was able to get a band corresponding to the lenght of my GOI leading to the assumption that the ligation was a success. 

I do use 100 microL /ml of amp, sorry for that. For the bacterial strains, I already use TOP10 (invitrogen), DH5a, and JM109. The DH5a and JM109 were both prepped in the lab while TOP10 was purchased. Yet, all failed to produce transformants. 

I have done the heat shock for 30 sec and 50 sec but both failed. I even maximize the incubation on ice at 30 min (initial) and 10 min (after heat shock). Add 250 microL of SOC medium to the transformation reaction and incubate at 37 deg C for 2H. I plate 50 microL and then collect the cells by centrifugation at 5000 xg for 5 min. Remove 200 microL of the media, and plate the remaining. Before, I thought that maybe the L-rod was too hot when I do the spreading, so I became mindful of that. But still my transformation are failing.

Hope I get more insights from you. Your answers are really helpful!

It seems that there is a problem in antibiotic concentration. Did you use the same LB plates with antibiotic concentration as your lab mates? 

Use the method, documented exactly as below, i have used this method without fail and it has given good colonies on plate always. Make lb broth of two kinds, 1 with ampicillin and one without ampicillin (conc 100 microlt per ml) .

For LB broth (without ampicillin) : take 1 gm of LB powder mix it with 50 ml autoclaved water and autoclave it and keep at 4 degree fridge.

For LB broth (with ampicillin) : take 2 gm of LB powder mix it with 100 ml autoclaved water and autoclave it and keep at 4 degree fridge and add ampicillin in above  mentioned concentration

Make LB Agar : Take 6 gm of LB powder mix it with 200 ml autoclaved water and autoclave it and add ampicillin in above mentioned concentration, along with a color substrate like IPTG XGAL and pour the plates when the when this mixture comes to 46-48 degree temperature. Seal the plates with parafilm. Keep them inverted in a stack and cover with silver foil at 4 degree fridge.

Now take your comp cells, thaw them n flick a little. Now add the comp cells gently to 1.5 ml mct and add the ligation mixture containing your ligated plasmid. Leave it on ice for roughly half an hour. After half hour, put the tube in a float and put in a water bath pre set at 42 degrees for exactly 1 min. Now immediately put this tube in ice for 10 mins. Now add LB Broth  900 microleter(without Ampicillin, as at this stage comp cells are very sensitive). Put this tube gently without inverting in a shaker incubator at 37degrees for atleast 2 hours. 

After 2 hours, check the tubes for little turbidity, now centifuge the tubes 5 mins 8000 rpm at RT and decant the Broth.

GO TO THE LAMINAR HOOD NOW

Now, add 50 microleter of fresh LB Broth without ampicillin and resuspend the pellet. Spread the cells on previously made Agar plates (with Xgal- iptg/Ampicillin ), seal them, keep them always inverted and put in a stationary incubator at 37 degrees overnight.

Make, sure that your plates and LB Broth are both at room temperature before use. otherwise the bacteria will get a heat shock. Next day pick white colonies and inoculate in LB Broth (with Ampicillin) and proceed ahead.

Kindly,do it an di hope your problem will be solved. Please tel what the results were like after you used this method.

Thanks for your patient reading...

Shivani

There are a few problems that catch my eye in your protocol. FIrst and foremost, never keep your competent cells on ice for such a long time before use. A few minutes on ice should be adequate to thaw (2-3 min), and then proceed immediately with your transformation.

Second, make sure your heat shock is at 42 exactly, using a water bath (not a heat block or incubator). Time of incubation is critical. Optimal timing can vary with strain and prep, so I would try three separate transformations using a positive control plasmid (supercoliled, NOT ligated at around 1ng total) for 20", 30", and 45" initially.

Third, after heat shock, return to cell to ice for exactly 2 minutes. Use SOC at RT. Some protocols call for 37C, but I wouldn't recommend it for homemade competent cells. 

For AmpR, 30-45' recovery at 37C is adequate (not 2hr). Shake at 220rpm or less (faster speeds will harm your transformants).

Finally, check that your LB plates are good. If the pH of the media is wrong (too acidic) or a contaminant is present (e.g., detergent from unclean glassware, or accidental contamination with bacteriophage), your bacteria will never grow. Try LB plates that someone else has validated. If Amp was not included when the plates were poured, you can always spread it on top prior to plating your transformation.

Until you get encouraging results using a positive control plasmid, you shouldn't try to troubleshoot your transformation with the ligation mix.

If you have commercial grade, heat-shock competent TOP10 cells, give them a try. If those fail with the provided positive control, then there is definitely something wrong with your SOC or your LB plates.

thank you for all the response. I will take all of them into consideration. will do transformation tom. hope it works! 

 Hi everyone.

So I redid the ligation and transformation. My transformation using a whole plasmid worked! However, the transformation for the ligation reaction failed to produce any colonies. I can clearly say now that the problem might really be in my ligation.

This will be a totally different question, but is there anyone who has an experience in using pET 101 D-TOPO vector by Invitrogen? Could anyone share their protocol for ligation using the said kit? TIA

What do you think made the difference for the positive control? Linear DNA has about 10% the transformation efficiency compared to supercoiled DNA. In addition to that 10-fold hit, not all the plasmid in your TOPO reaction or a standard ligation will undergo recombination, so you should expect anywhere to 50-100 fold fewer colonies than with an equivalent amount of supercoiled vector. That is why for most subcloning or TOPO reactions, competency of 10^8 CFU/ug DNA or better are highly recommended.

Daniel,

Thank you for your response. Therefore, a freshly prepared CCs would probably address my problem on transformation? Antother question though, will increasing the CC volume used per tansformation reaction increase my chance of observing a transformant?

TIA.

Fresh competent cells might help, as will using more bacteria, and plating a larger fraction (or entirety) of the transformation reaction. If you are performing TOPO cloning, it is essential that you do not have excess insert in the reaction. 1:1 molar ratio of vector to insert is ideal. If your insert is small and/or concentrated, you may need to dilute it after PCR. For T4 ligase-based cloning, use a 1:3 molar ratio.

I forgot to mention that I usually plate the whole transformation reaction. Also, the pET 101 D-TOPO kit does not use T4 ligase. Ligation reaction is only composed of the pcr product, topo vector, salt solution and water. according to the manufacturer, ligation can occur in just 5-30 minutes at room temp. Did anyone experience or used this specific vector before? How did you modify/optimize the ligation reaction and transformation? 

TIA

I've used the TOPO system many times, albeit different TOPO vectors. Follow the manufacturer's guidelines. Make sure you have a single PCR product (if not, gel isolate the correct one). If your template DNA is also an AmpR plasmid, you need to get rid of it by DpnI digestion or by gel isolating your PCR product. Once you have purified your PCR product, check its concentration by UV absorbance, and add 1 molar equivalent to the TOPO reaction. 5' incubation is adequate for inserts

 TOPO vectors lose activity with storage. Make sure your cloning kit is not too old.

Hi Daniel,

Thank you for your series of answers! They've been very helpful! I was actually thinking that the TOPO vector might really be the one at fault. The vector that I am currently using is actually a hand-me-down from a previous student in the lab. It's probably around 2 yo and was stored in the freezer. 

Hi grace, I hope you have overcomed the problem, I actually experience the same problem like yours, I use same invitrogen PET 101 D/TOPO kit. I totally agree with Dr Daniel, topo vector might be the problem. We bought a new kit and it works perfectly now