I am trying to clone my PCR products, but I end up with immense amounts of false positives. Only between 2 and 50% of the white colonies actually contain an insert. So what could be going wrong?
This is what I do:
First I do a PCR on my DNA sample, resulting in multiple bands on agarose gel. The desired fragment is cut out of the gel and solved in 1X TE pH 8,0 for 2 days. Using this 'product', a new PCR is set up, using the same primers as in the first PCR, resulting in an intense band of the desired fragment on agarose gel. All PCRs are performed using Amplitaq polymerase, to create the A overhang and have a final extension step of 7 minutes. I clean the PCR product using a Macherey-Nagel system. Here I bring the PCR product on to a membrane and wash away the contaminants (dNTP, salt, dimers, ...) using MQ and vacuum. Afterwards I extract the DNA and solve it in MQ. The concentration is measured using a Nanodrop and checked on agarose gel. Since the concentrations are very high, I make a dilution in MQ so I can add somewhere between 1 and 3 µl in the ligation step.
For the cloning, I use the pGEM-t system (not the easy kit!).
The ligation needs a certain amount of DNA. I calculate this using their formula:
(50 ng * x kb insert)/ 3kb = a
a * (3/n) = b
b/ concentration PCR product = c
c = volume (µl) of PCR product that has to be added in the ligation step.
n depends on the size of the insert. For inserts of 500 bases or less, n = 1, for inserts of about 700-800 bp, n = 2 and for inserts of 1500 or more, n = 3.
Using this formula, I add somewhere between 20 and 25 ng of DNA in the ligation reaction.
I set up the ligation in the evening, around 9 pm. The mix (5µl 2x ligation buffer, 1µl vector (50ng), 1µl T4 ligase, x µl PCR product and add MQ to final volume of 10 µl). The ligation stands at room temperature (20°C) untill 8 am. Then I put it in the fridge (4°C) untill 2 pm. This provided other colleagues the best results in the end.
At 2 pm, I start the transformation, using JM109 competent cells. The water bath is exactly 42°C and the mix is only put in the bath for 45 seconds, after which it is immediately returned to ice. The shaking at 37°C is done for 90 minutes and the temperature of the shaker is checked using a traditional alcohol thermometer. I plate out 100 µl and 200 µl in triplicate, using a Drigalski and incubate overnight at 37°C.
In the morning, plates are taken out the 37°C and put in the fridge untill 2pm, after which I select white colonies and put them on a new plate.
Here, the problems start. I have 4 sizes of insert I want to clone: 360 bp, 500 bp, 800 bp and 1500 bp. The longer the fragment, the more white colonies I see on the plates and the less blue there are. For the 1500 fragmentn, I obtain around 300 white clones that I can put on a new plate, leaving about 150 blue clones. For the 360 bp fragment however, I only obtain 11 clones (white + light blue). The rest of the plate is filled with blue colonies (±200/plate). The other fragment sizes are somewhere in between there. For the 500 bp fragment I picked up 200 clones and left 800 blue ones. For the 800 bp fragment I picked up 175 and left 500 blue ones. So actually something probably goes wrong there, since I should get around 80% white clones according to Promega. However, one of their employees told me everything works fine since I do get that many white clones. Only for the smallest insert they did not have any explanation, except maybe the insert: vector ratio. They told me to 'play' with that ratio, since it can vary from a 16:1 to a 1:8. But I'm not quite fond of doing this, since cloning is a costly and time consuming proces.
To continue, after I select white colonies, I put them on a new plate and incubate them at 37°C again untill the next day.
What I do notice the next day, is that some clones turned dark blue, other light blue. The smaller the fragment, the more clones turn to light and dark blue. I pick up all left over white clones and the light blue ones and put them in 15 µl of MQ, after which I boil them for 10 minutes and centrifuge (just following the protocol).
Ok, so now I have my clones and I start checking if an insert is present in the ones I picked up. I use the ELRNA55 PCR with the SP6 and T7 primers. Here, the big problems start. After PCR, I put 5 µl on agarose gel. And I notice a lot of the clones do not contain an insert. For the 500, 800 and 1500 bp fragment, I PCR'ed 95 white clones, resulting in 33, 4!! and 51 clones that actually had the correct insert. The other clones were either empty, gave me a an insert of a smaller size (10% of the cases) or gave me 2 bands at a position of ±200 and ±400 bp.
I have contacted Promega and they tell me everything seems to be working fine. We went over every single step from the start. They asked me to set up 4 different controls the next time I clone:
1) background control: no insert added
2) positive control: using the supplied control DNA
3) transformation control: Check the transformation efficiency of the competent cells by transforming them with an uncut plasmid (not pGEM®-T or pGEM®-T Easy, since these vectors are linearized) and calculating cfu/μg DNA
4) ampicillin control: plate competent cells ons plate with and without ampicillin
I have been thinking about using a different kit, but than again I wonder if that will give me better results, since the general protocol is the same (ligation, transformation, ...).
If the problem is the insert:vector ratio, how can I find out if that is the problem and what ratio should I use to get more clones actually containing an insert? Because thesting different ratios isn't really an option. I have read several topics where people use a higher ratio, especially 3:1 (and add somewhere around 75-100ng of DNA). I also found that people tend to use a higher ratio if the fragment is smaller. So for a fragment of a size smaller than 300, a 5:1 to 10:1 ratio is used. But I don't want to add too much DNA since that will probably inhibit the reaction?
Could degradation of the A overhang have happened? And how can I solve this? Just incubate my cleaned PCR-product at 72°C for 15 minutes in buffer, dNTPs and taq and clean it again afterwards?
Since I want to obtain a decent amount of clones, I'm quite stuck here, since I get that many false positives. Hopefully someone here can help me or get me on my way. Because now I lose a lot of time and money.
Thank you in advance!
Blue/white selection sometimes doesn't work for very short inserts if they occur to complement the ORF of the beta-galactosidase. For the 360 bp insert try to analyse also some blue colonies, just in case....
There are a few things that you can try to improve your yield.
1.- Try a new kit, perhaps the old one has lost the overhangs. I've been using pGEM-T for years and it is very robust, but any nuclease contamination will get rid of the T's
2.- Perform the ligation at 12-14ºC overnight. It is enough, T4 DNA ligases work very well at this temperature, and you are reducing any contaminating activity (nuclease) that would feel happy at 20ºC. If your ligase is old, change it. Change also the ligation buffer because it is made with rATP that is pretty labile.
3.- Try making mini-preps of your white colonies. I know that it is harder than PCRing them, but a miniprep gives you more information, specially if the cloning didn't wok (is there plasmid? which size? larger than the control?)
And a final word about cloning.A succesful cloning (even for pGEM-cloning) is that one that gives you AT LEAST one clone with the expected insert. Cloning is not a contest on how many white colonies you have, and you don't have to be stuck because you yield is lower than the stated by PROMEGA. You just need a colony with insert, and once you've got it (even if it is the only one in the whole plate) carry on...
With a lot of false positive colonies after ligation I would always suspect unspecific religation of your plasmid. I don't know this kit, how comes the plasmid? Do you digest it yourself or is it provided in a digested form (probably even phosphatase treated)?
The other question is: Which antibiotics do you use for selection, at which concentration and how old is your stock?
Otherwise I really recommend doing the controls that the Promega service guys suggested. I enclose at least the first two in every ligation I do, so I know for sure, whats going on.
Hello Christian,
Thank you for your reply!
The vector from the kit normally comes in a digested form. The link below gives you the information about the kit I use:
http://be.promega.com/resources/protocols/technical-manuals/0/pgem-t-and-pgem-t-easy-vector-systems-protocol/?origUrl=http%3a%2f%2fwww.promega.com%2fresources%2fprotocols%2ftechnical-manuals%2f0%2fpgem-t-and-pgem-t-easy-vector-systems-protocol%2f
As for the antibiotics, I use ampicillin. I start from a stock of 100mg/ml. The ampicillin comes in powder form and when I prepare the mdium, I make the 100mg/ml stock in MQ, using a cellulose filter. The final concentration in the plates is 100µg/ml.
The ampicillin stock is quite old I have just found out. The person responsible is looking up when it was bought. We believe it is somewhere around 4 years old. However, the same ampicillin was used by someone else in the lab in November and it gave this person good results. With good I do not mean optimal. That person also plated out 100µl and 200µl in triplicate and this gave them somewhere around 200 white colonies in the end (but also 200 or more blue ones). All the white colonies did have an insert.
On Sunday, I will redo the cloning experiment and include the positive control, background control and ampicillin control. To be sure, I will use new ampicillin from another group in our lab.
As for the controls, in the past we always included them and everything always worked fine. Because of that the controls were not enclosed anymore because of the extra cost. However, I will enclose them again to avoid further problems.
Many thanks again for your answer!
If the vector comes digested, I think this should be ok. The same is true from Ampicillin if the powder is stored in the fridge. The problem can be stock solutions, which have undergone too many freeze-thaw cycles. Also the concentration seems to be fine (if its too low then falso positive satellite colonies can occur).
And you know Murphy's law: As long as you enclose the controls, everything is fine...
I know that it takes additional costs and effort. Lets see, how the controls look like.
Blue/white selection sometimes doesn't work for very short inserts if they occur to complement the ORF of the beta-galactosidase. For the 360 bp insert try to analyse also some blue colonies, just in case....
There are a few things that you can try to improve your yield.
1.- Try a new kit, perhaps the old one has lost the overhangs. I've been using pGEM-T for years and it is very robust, but any nuclease contamination will get rid of the T's
2.- Perform the ligation at 12-14ºC overnight. It is enough, T4 DNA ligases work very well at this temperature, and you are reducing any contaminating activity (nuclease) that would feel happy at 20ºC. If your ligase is old, change it. Change also the ligation buffer because it is made with rATP that is pretty labile.
3.- Try making mini-preps of your white colonies. I know that it is harder than PCRing them, but a miniprep gives you more information, specially if the cloning didn't wok (is there plasmid? which size? larger than the control?)
And a final word about cloning.A succesful cloning (even for pGEM-cloning) is that one that gives you AT LEAST one clone with the expected insert. Cloning is not a contest on how many white colonies you have, and you don't have to be stuck because you yield is lower than the stated by PROMEGA. You just need a colony with insert, and once you've got it (even if it is the only one in the whole plate) carry on...
Hi Estanis,
Thank you for your reply.
For the 360bp fragment I have been thinking about that in the past. However, I have picked up several blue colonies (approx. 5) and performed the ELRNA55 PCR on them. Sadly, none gave an insert.
The kit is quite recent. Competent cells were bought only 3 weeks ago. The other components (except buffer) were bought in November and should work fine. The buffer however is a lot older. However, this same buffer was used by another person in the lab a while ago (see above comment). Yet I have been thinking about just buying a new kit to rue out contamination or degradation of the components. This will probably be the easiest way.
I will perform the ligation at 15°C or 4°C next time, overnight, hoping for better results.
The Miniprep sounds like an excellent idea, I will keep that in mind in case the cloning experiments keep failing.
I know one clone is a result, but if you are looking at the abundance and diversity of a certain gene in a sample, like I am, the more clones you get, the better. Because with 4 succesful clones out of 95, I can not do a lot. And it would take me a lot of time and money in order to obtain enough clones. But this is research I suppose, sometimes it works, sometimes it doesn't. I always try to keep in mind what Edison said: I have not failed. I've just found 10,000 ways that won't work.
A little tips, do remember to add the ampicillin till the temperature of the medium is lower than 55 °C (touch the bottle and feel it ). And for more directions, please remember there is an important book, Molecular cloning: A laboratory manual.
Hope it will help. Good luck!
To give the people that already helped me an update:
Sunday I set up a new ligation around 8.30pm, including the controls Promega supplied me with. Everything was put at 4°C overnight.
Yesterday at 2pm transformation was done. All times and temperatures were checked. Water bath and incubator temperatures were checked with alcohol thermometers and devices were switched on at 10am to make sure temperatures were stable.
For my genes of interest, I now added 3µl of DNA instead of using the supplied formula. For both of my genes this resulted in a ratio insert:vector of 14:1.
Leftover competent cells were dissolved in 300 µl SOC medium and 100 µl was plated out on a plate without ampicillin, a plate with new ampicillin and a plate with old ampicillin.
For both positive and background control, 100 µl was plated out in duplicate.
For my genes of interest, 100 µl and 200 µl were plated out in triplicate.
Around 5pm everything was plated out and plates were incubated untill 9am this morning at 37°C, after which they were put at 4°C untill 2pm.
Results:
Background control: one plate with 9 and one plate with 6 blue colonies.
Ampicillin control: plate without amp. is filled with colonies. Plates without amp. are empty.
Positive control: one plate with 44 and one plate with 61 colonies, all blue
Own genes: ±50-100 colonies per plate,
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