How much ligation reaction for transformation

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You may have used an incorrect part in your reaction, thus resulting in an incorrect antibiotic backbone. Incorrect concentration of antibiotic: Make sure you use the correct amount of antibiotic in your plates. See Antibiotic stocks for iGEM's guidelines for the correct concentration of antibiotic to use for the BioBricks plasmids.

Excessive freeze-thaw: If you are using competent cells that were thawed, re-frozen, and thawed again for transformation, you will see a large decrease in efficiency up to a two-fold drop in efficiency. It's best to use competent cells that have never been previously thawed for best results.

Too Many Colonies or a Lawn of Growth Another problem you may encounter is having far too many colonies that result in a lawn of growth on your plates. Your first reaction may be to think that this is a good result, but often this result indicates a problem. Below are some common problems that can result in a lawn of bacteria growing instead of single, isolated colonies after transforming your DNA.

Plasmid transformed into highly competent cells: If you have a high transformation efficiency and you transform plasmid, you can sometimes get a lawn of cells growing. There's an easy solution to this problem: plate fewer cells after the recovery step in your protocol. This will dilute the cells across the plate and should result in isolated colonies. Adding antibiotic to hot agar: When making plates, you need to make sure the agar is cool enough for the antibiotic to be added while still being molten.

If the agar is too hot when you add the antibiotics, you can breakdown the antibiotic and thus make plates with little to no antibiotic present. Improperly mixed plates: If you make your plates without the aid of a stir bar to help mix the antibiotic into the agar, you can pour plates that have an uneven amount of antibiotic in them. Likewise, if you spread plate antibiotic onto your plates after they harden, you need to make sure the plate is properly covered.

Patches of dense growth can result from improperly mixed or spread plated antibiotic, making it difficult to select an isolated colony. Lack of antibiotic: You may have forgot to add the antibiotic to your plates or you may have used a plate without antibiotic by mistake. A lawn will result if you use a non-selective plate because the E.

Possible Protocol Problems Below are some other considerations to take into account when troubleshooting your transformation protocol itself. These are the common steps in a chemically competent protocol that can be easily altered to attempt to improve your transformation results.

This often helps increase the number of colonies you'll see on your plates. Heat shock: If you follow a chemically competent protocol, heat shocking your cells is often a part of your transformation protocol. Depending on the type of tube you use, you may need to alter your heat shock parameters. For PCR tubes and other thin-walled tubes, a shorter heat shock time of 45 seconds may be fine. For microcentrifuge tubes or other thick-walled tubes, I would recommend increasing the time to seconds.

Recovery Time: If you're still seeing a low number of colonies, you can also try increasing the length of your recovery time for your cells. Transformation Troubleshooting One of the most common problems that researchers have in the lab is transforming their DNA successfully into E. Transformation Efficiency Kit.