Transformation and miRNA
Simoe Goss
Wednesday, 04 February 2009 23:10 UTC
Hello,
I am a Master student and my project involves miRNA research.
My biggest hurdle so far involves transformation.
I seem to be struggling to get any colonies to grow on my agar plates.
I am using a pGL-3 control plasmid, where I inserted my miRNA target sequence using a single restriction cutting site using XbaI.
I added ligation reaction to the DH5-alpha cells. I than put the reaction on ice for 30 minutes, than heat shocked it for 45 seconds at 42 degrees Celcius.
After that, I added LB and left the bacteria to grow overnight at 37 degrees Celcius. I than spread the colonies onto the agar plates.
I use 100 microlitres of Ampicillin/1ml of 1.5x Agar with LB
I use 3 controls, one control without the oligo/primer, positive control (using uncut pGL-3) and negative control (cut pGL-3).
The results I have been getting is as follows:
- postive control shows a few colonies, other controls no growth and no growth for the miRNA target gene
or
- no colonies on either plates including all three controls
Can someone please give me advice on this? It would be extremely appreciated
I would also like to state that I have tried various competant cells including DH5-alpha (fresh and frozen) and JM109 cells, and ordered fresh Ligase, for the ligation reaction.
Thanks.
Updated 04 February 2009 23:19 UTC
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Replies
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Anonymous
Are your competent cells homemade? You could consider using commercial cells with high efficiency.
And what’s the concentration of your Amp stock? 100ul/1ml medium sounds a lot to me, usually it’s 1000x (so you take 1ul/1ml).
Good luck. -
“postive control shows a few colonies”
That might be one problem. There should be many colonies for the positive control, not a few, if it’s just the uncut plasmid that you’re transforming. I would first make sure your transformation is working really well before you even attempt the ligation again. Try a different batch of plasmid, or more, or cleaner, or a different plasmid that also has Amp resistance, just to see what conditions you need to get a plate with LOTS of colonies for the control.
As for the ligation itself, you can try different ratios of insert:plasmid. (But first get the transformation of the empty plasmid working!)
I’m also a bit worried about using only one restriction site, but that would give you false positives – not empty plates. (Because it might ligate back on itself without an insert in it.)
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To Anonymous, I apologise for that type error.
The Ampicillin concentration is 100mg/microlitre. And yes it is home made.
I add 800 microlitres of Ampicillin to 400mL of LB. Than pour it into the dishes. So approximatly 40 microlitres ( at a concnetration of 100 micrograms/ml) per agar plate. -
Hello Eva,
Thank you for your reply.
The latest transformation I did, showed no colonies for the positive controls.
I used DH5-alpha cells (my batch), DH5-alpha cells from my college and tried using JM109 cells. None of the positive controls showed any growth.For the ligation I have tried different ratios of inserts and plasmids, but I still get no colonies.
I have tried different batches of Plasmid, but maybe consideringa different type of plasmid with amplicillin resistance might be a good idea.
Thanks for your advice
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sorry Anonymous I meant 100 micrograms ampicillin /1ml. Sorry I am half asleep.
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Forget about optimizing ligation until your positive controls work… It might be working – you just can’t tell, and you’re only wasting reagents!
I don’t know what could be going wrong… Did your transformations work before? Is anyone else in the lab using the same batch of LB plates (and are their transformations/cultures fine?) because it could also be the plates.
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The reason could be anything..but as per u experience.
i would really check the competent cell first, using different plasmids.
for an uncut vector u will found uncountable no of colonies even at 50 ng of of plasmids used. untill u will not get this condition, atleast don’t try to transform the ligation mix. Check the different lots of comp cells, even u can borrow from someone who have recently used the one and working well. So first standardize ur transformation and then move further..
u can also do plate the only comp cells on LB plates without any antibiotics to check whether cells are viable or something is wrong…don’t forget to reviwe the cells in lm medium at 37 C for an hr.Regards and All the best….
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Simoe,
What volume of LB are you innoculating and incubating overnight? Is this cloudy in the morning before you streak the plates? I havent done this in a while but I think instead I put the cells in SOC medium for like an hour at 37 degrees and then streak the plates. I dont think you need to grow them overnight and it could be possible that they are all dead because they overgrow and run out of nutrients by the morning if they are in a small volume.-Jason
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Thank you very much for all the people in this forum and the advice they have given me.
I have discoverded what went wrong so far.
After doing a multitude of trouble shooting attempts I came up with the following:1. The DH5-alpha and the JM109 cells I used were not competant.
I did a positive control test using my DH5-alpha and JM109 cells,against other batches of DH5-alpha cells provided by two of my colleges.
2. I added 10 microlitres of ligation to 25 microlitres of my colleges competant cells and did the transformation
– 15 minutes on ice
– heat shock at 42 degrees Celcius for 60 seconds
– put the samples on ice for 2 minutes
– added 1ml of sterile LB to the sample
– put the samples in a shaker for 1 hour at 37 degrees Celcius
– spread them on a agar plate with Ampicillin and left the plates overnight at 37 degrees Celcius.
The result was I still had no colonies2. So the next step, I ordered new ligase enzyme and made a new ligation mixture.
I compared the old ligation mixture and the new ligation mixture and did a transformation using my colleges competant cells, and repeated the same transformation method I described above.
I also included a control (no oligos), negative control (cut vector) and a positive control (uncut vector).The next day I saw hundreds of colonies on my plates using the new ligation mixture and no colonies with my old ligation mixture (including the positive control).
So it was a combination of both non-functional ligase enzyme and cells which were not competant.
I will now isolate each colony and grow them in 5 ml of LB with 10 microlitres of ampicillin (100 microlitres/ml)and will incubate them over night at 37 degrees Celcius in the shaker and after that I will do a mini prep to purify the DNA. I will than take a small sample and do a restriction digest and do a gel electrophoresis on 2% agarose gel and hopefully I will see some inserts.Since my inserts are small (around 25bp), would it be wise to use 3 or 4% agarose gel to detect the oligos?
Thank you very much.
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I used the Qiagen Spin Mini prep (250) kit to purify my DNA after my transformation. I made around 30 samples, at the end I got rather low levels of DNA with an average of 22ng/microlitre with a total volume of 40 microlitres.
Is their any technique to increase the DNA yield?
And lastly, I checked if the oligos had inserted into the vector. I used 17 microlitres of each sample and added 1 microlitre of restriction enzyme and 2 microlitres of Buffer H.
When I ran a gel on 3% agarose/TAE buffer using ethidium bromide, no bands showed up for the oligos and the vector bands were barely visible.
Could someone please give me some advice on this?Thank you very much!
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