A science question

[lwong]

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Hey yall, once again I'm throwing in a science question as a novice undergrad researcher... 🙂

I've been looking at some E.coli transformation protocols and I have found some conflicting incubation time on ice as well as heat shock times.

For Incubation on ice:
so far I have seen times ranging from 5-60mins? how do I decide how long I should incubate for? I guess what's going on at this process (more specific than that the DNA are getting into the cells)?

For heat shock times:
I've seen some that recommend seconds and those that recommend minutes. Some even don't include this step. What exactly is going on when I subject the sample to a heat shock?

 

[Nuel]

I have no practical experience with bacterial incubation times and transformation.

Heat shock would be when you allow your circular DNA plasmid to penetrate the bacterial cell membrane. You should check with a postdoc, grad student or someone in your lab to help you with timing. Based on my theoretical knowledge of the subject (classes and all), you may to incubate for 10 or 20 minutes on ice since leaving the cells much longer could induce damage as a consequence of too much thawing. Check with your lab. Heat shock time is usually at half an hour in appropriate media. Ideally, you would want to incubate long enough to allow all plasmids penetrate E.coli cells. Check with your lab again. Also see the book Molecular Cloning by Joseph Sambrook--your lab should have it.

Regards

 

[SaltySqueegee]

I too have mostly book knowledge on the subject, and have only transformed e. coli twice in the distant past.

As you are possibly aware, there is more than one way to skin a cat. As is the same with transforming protocols. You may want to see what your lab's specific protocol is. The reagents used can vary from kit to kit, and hence the varying in the different times for the various stages you may have heard of.

Your lab should have a cookbook of their most commonly used protocols; check that. Also, check with a fellow lab mate.

Regards,

-Salty.

 

[ClarinetGeek]

The above advise is great!

This is one of those procedure where everyone has a different technique and various reason for using it. To a certain extent, I would imagine it would be dependent on the strain of e. coli and DNA sample.

I have ton more transformations than you can shake a stick at. Generally, I incubate on ice for 20 mins, and heat shock for 2 mins. After which, I will add some rich media and shake for 1 hr at 37 C before continuing with the next step, usually spreading on an antibiotic selective plate.

 

[Doctor&Geek]

We just switched from company-purchased E. coli competent DH5a cells to homemade competent cells. Transformation efficiency was decreased 10-fold by using a 37 degree heat shock for 45 seconds using our homemade cells versus using a 42 degree heat shock for 60 seconds, but the company cells suffered a decrease in transformation efficiency at 42 degrees.

Bottom line - you'd be better off optimizing the protocol yourself if you're using homemade cells.

 

[b&ierstiefel]

Using homemade competent cells, I would incubate the bacteria + ligation mixture on ice for 15 minutes. Then I would heat shock at 42 C for 1 minute followed by reincubation on ice for 5 minutes (to help bacteria recover from the heat shock). I found this worked fine.

I've also used these really kickass ultracompetent cells made by Invitrogen. Incubate on ice for 30 minutes, heat shock at 42 C for 30 seconds, another 2-5 minutes on ice.

For either case though, transforming bacteria with supercoiled DNA (i.e., a miniprep DNA which you just want to make more of in a maxiprep) is going to give you significantly better transformation efficiency/colonies than transforming bugs with ligation mixture. That's because a plasmid created by ligating an insert to a previously cut plasmid using T4 DNA ligase in a tube is not going to be supercoiled. Supercoiled DNA has a much easier time entering competent cells than non-supercoiled DNA because supercoiled DNA is more compact in size.

 

[cmb81]

The heat shock period is dependent on the type of cells your using for transformation. I have used DH5alpha, BL21, and TOP 10 F' cells. The temp. is usually 42C for 30-45 sec then put on ice for 2 min.

FYI -- I always get alot more colonies when i use TOP 10 F' cells.

 

[dr.z]

After I thaw my bacteria, I incubate with DNA on ice for 30 min.

I heatshock them for 90 sec at 42 degrees.

Then incubate on ice for 2 min.

I used to leave it at 37 degrees for an hour, but I don't do it anymore. I still get pretty good efficiency.

 

[lwong]

Thanks yall for all your insights! I'm testing out the protocols we have for our homemade cells, and trying to make it more efficient. Actually, it's more like getting it to work in the first place. My group has been getting some suspicious results. For instance, the plate w/ the cut, but had not dephosphorylated vector, which I expect to rejoin, had about the same # of colonies than my uncut. In another instance, others in my group, after a quick and dirty gel analysis of the sample had only found a plasmid about the size of 1700 bp (the plasmid we wanted is about 5800 bp) We kept our total concentration less than 5microgram/ml, which is recommended by NEB and textbooks. Any suggestions to what's going on?

Oh yeah, does anyone have any suggestions to getting rid of the condensation on in the drug plates, which is stored in the fridge?

 

[dsq]

to get rid of condensation, warm the plates in the incubator for a little while before using them. lids off, and plates up-side down.

 

[lwong]

to get rid of condensation, warm the plates in the incubator for a little while before using them. lids off, and plates up-side down.

I tried warming the plates in the incubator at 37 for a little bit, but I didn't leave them in too long, afraid that the drugs wouldn't be able to survive. How long did it usually take for your plates? Also you had no problems with contamination since you take off the lids?

 

[dsq]

since you will probably be incubating the plates at 37 anyways, warming them for a little bit before plating shouldnt hurt the antibiotics. Just make sure that the open side of the plate is facing down. I have never had a problem with contamination doing this. I usually warm them for 30-45 min, then plate transformed bacteria.

 

[logos]

To get the condensation off of the plates, I just take off the lid and bang it against a paper towel. This is "genetics sterile technique" where you dont have to be extreamely carefull because you have antibiotics in the medium (optimally double selection -- 2 diff. antibiotics).

As far as the transformation protocol, I would use the one from the same source as you got the protocol for the competant cells. (The two really famous lab manuals are "Current Protocols in Molecular Biology" and "Molecular Cloning" according to my former PI, so those were the ones we used)

Just make sure the heat shock part is not too long or two hot. The longest I think that I have heard of is ~1min but there could be some protocols for longer. The period after that with the ~1ml of media shouldnt be too long. You want enough time for the cells to begin to recover, but not enough time for them to begin replicating, as it does you no good to have several times more colonies than you need, with each transformant being represented by many colonies.

Good luck!