The polymerase chain reaction (PCR) works great for screening vectors for insert. You just have to make sure the PCR primers you use flank the multiple cloning site (insertion site) that contains your sequence of interest. When your insert is PCR amplified and run on a gel, remember that the amplicon will be a little larger because some of the vector will be included depending on where you chose the PCR primers. If the amplicon is not larger than your insert, it may mean that particular clone has a truncated insert. I have typically picked a colony with a toothpick, swirled it in 10-20 ul sterile nuclease free water and then dropped the tooth pick into 1.5 ml broth culture (all done aseptically). You boil the colony you transferred into the water for 5 minutes (in a PCR machine) and then use 1-3 ul of that for your PCR reaction. In the meantime incubate the broth cultures you inoculated. The next day you run the gel for the PCR reactions and pick the corresponding broth cultures that have suitable insert for extraction expression or whatever you happen to be doing. This screening protocol works well for both small and large scale screening efforts. I have used PCR screening for inserts in 8 well, 96 well and 384 well formats.

The polymerase chain reaction (PCR) works great for screening vectors for insert. You just have to make sure the PCR primers you use flank the multiple cloning site (insertion site) that contains your sequence of interest. When your insert is PCR amplified and run on a gel, remember that the amplicon will be a little larger because some of the vector will be included depending on where you chose the PCR primers. If the amplicon is not larger than your insert, it may mean that particular clone has a truncated insert. I have typically picked a colony with a toothpick, swirled it in 10-20 ul sterile nuclease free water and then dropped the tooth pick into 1.5 ml broth culture (all done aseptically). You boil the colony you transferred into the water for 5 minutes (in a PCR machine) and then use 1-3 ul of that for your PCR reaction. In the meantime incubate the broth cultures you inoculated. The next day you run the gel for the PCR reactions and pick the corresponding broth cultures that have suitable insert for extraction expression or whatever you happen to be doing. This screening protocol works well for both small and large scale screening efforts. I have used PCR screening for inserts in 8 well, 96 well and 384 well formats.

I agree with Michael- use PCR colony screening. However, you should always run a negative control ligation (vector, but no insert) to get a sense of your cloning efficiency. If the # of colonies on the no insert control plate is similar to the vector+insert condition, then PCR colony screening won't help much because you are unlikely to find a positive clone. In practice, you should expect to see at least a two fold difference in the number of colonies on your + vs - insert plates.

Thank you so much for your suggestion.

However, my lab has never done this kind of screening before.

Thus, could you please confirm some points below:

1. I do PCR with the colonies directly, and do not need to extract the plasmid which will be used for PCR?

2. The primers used for that PCR are the same primers I used to get the insert, or I have to design new ones?

Thanks again!

There are a ton of variations on PCR colony screening, but none require isolation of the plasmid in advance. Simply resuspend your bacterial colony in 100-500uL of water and take 2 microliters of this suspension as the template for your PCR reaction. Make sure you heat your PCR + template mixture for at least 5min at 95C before proceeding with the normal cycling parameters.

You might be able to use your primers from the original PCR reaction for screening (I often do). The basic requirements are that your primers must NOT recognize any targets in the E. coli genome (you can BLAST to check, but almost never an issue for eukaryotic cDNAs), and preferably the Tm of your primers should be 55C or higher (to avoid non-specific target amplification).

Thank you so much Daniel.

And one more ask for help, do you know any good protocol (online or somewhere else) for that PCR process?

I greatly appreciate for that.

@Leo: Any standard PCR that works in your lab will work. Increase the initial denaturation to 5 minutes, use a bacterial solution as a template as described above and start over. I would recommend using a positive and a negative control and also a postive control with empty bacteria in it (to test for inhibition). Its really as simple a it has been decribed here.

Definitely do the controls :-) The priming sites cited for your vector are listed below. When ordering the primers remember the reverse priming sequence needs to be reverse complemented relative to the forward primer. In other words each primer has to have its 3' end facing in the direction of the amplicon.

GLprimer2 binding site 89–111

RVprimer4 binding site 2080–2061

http://www.ebiotrade.com/buyf/productsf/Promega/tm033.pdf

Thank you all so much.

I will try tomorrow :)

Dear Leo,

I agree with Daniel Cohen and I usually do a control for ligation : pGL3-basic vector digested with Ligase but without insert.

After transformation of ligations (with and without insert) I count the number of colonies present on the control to estimate false positives and I pick a proportional number of colonies from "real ligation" colonies. If you have few colonies in your control, even normal miniprep of 5-10 colonies may be sufficient for a good and positive clone!

Cross my fingers!

Hello,

The simplest way to confirm colonies containing your insert is to do colony PCR using a forward primer that binds to the flanking regions of the pGL3 vector close to your insert and a reverse primer that binds on your insert. Choose the primers in such a way that you would get a PCR product of say between 700bps to 2000bps. This is the simplest and fastest to confirm your insert.

After that you could go a step further by using appropriate restriction enzymes that produces two fragments upon cleavage of your recombinant plasmid, once you have first of all confirmed your insert using colony PCR. I guess this clue will be of help to you.

Wishing you the best of luck.

Greetings.

Osita

Hi all,

I performed PCR as your suggestions and get a good results. Some colonies have inserts and some do not.

However, I would perform enzyme cutting to confirm my first experience in this lab.

In your case, you just only use PCR for checking the presence of insert and do not use enzyme cutting?

Dear Leo,

I personally prefer to reconfirm positive clones by miniprep and enzyme cutting plus sequencing of insert and insertion sites. It's few hours more but it can be critic for the experiments you will perform with your construction!

Good luck

Dear Leo,

Like I informed you earlier, it is nice that you used colony PCR to sort out clones that possess your insert of interest. To pick the clones containing the insert in the correct orientation, you really need to do your restriction enzyme cutting, except, if you cloned in such a way that all clones containing your insert will be in the correct orientation. All the same, your restriction enyzme cleavage is necessary to confirm clones containing your insert in the right orientation.

Take care and best of luck!

Leo- since you are new to cloning, it is a good idea to perform the restriction digest as an additional confirmation, but don't stop there. The use of PCR amplification engenders some risk of introducing mutations into your promoter of interest. Thus it is essential that you sequence your insert. This is more important than simply confirming the presence of an insert using a restriction digest.

I only do colony PCR if the #colonies is similar the # on the negative control.

Best is to use one primer in backbone, and one insert, since otherwise you can have positive PCR even from a "no colony" pick (ligated DNA is all over the plate)

I always then confirm by running a restriction digest as well as uncut DNA on a gel. The uncut DNA removes the low frequency dimers that remain unnoticed during a regular digestion (I have seen several of those).