Troubleshooting Guide Red/ET Recombineering
Problems with the recombination reaction can be caused by a number of different factors. The following troubleshooting guide may be helpful in solving any problems that may arise.
For homologous recombination the two DNA molecules must share two regions of perfect sequence identity. The most common problems are therefore ‘wrong’ nucleotides in one of the homology regions, which will abolish recombination completely. Since oligonucleotides are used to add the homology regions they have to be synthesized properly and be of good quality.
Take into account that long oligonucleotides (especially if they are longer than 80bp) require additional purification steps. Please ask you local oligonucleotide supplier for the recommended purification. Homologous recombination will also not take place if the sequence of the BAC is not determined 100% correctly for the homology region.
If you are trying to target a repeated sequence in your BAC, you may experience problems because the homology region at the end of the linear fragment can go to more than one site. It is therefore best not to target repeats directly.
Observation: No colonies on your plate after Red/ET Recombination
Check the following parameters:
1. PCR product
The PCR product could be the wrong one (check it by restriction digest or sequencing)
The PCR product could be degraded (check an aliquot on an agarose gel)
The product could carry incorrect homology arms. Please double-check the oligonucleotides, which were used to generate the homology arms, for quality and correctness. If necessary verify the sequence by sequencing on the PCR product.
Insufficient amount of PCR product:
Increase the amount of PCR product from approximately 100ng up to 500ng. Please take into consideration that you may also increase unspecific background.
2. The plasmid or BAC
- could be the wrong one (check it by restriction digest). Make sure that you have the right BAC, make minipreps and characterize it by PCR and restriction digest. Some BACs are wrongly annotated, inherently instable or just a mixture of more than one BAC.
- could be degraded or instable (check an aliquot on an agarose gel or PFGE gel)
- the sequence which should be recognized by the homology arm is different or absent (check the presence by amplifying the region by PCR and sequence the PCR product if necessary)
3. The Red/ET reaction
- no expression plasmid present in the cells; e.g. the cells were grown at 37°C instead of 30°C (check for tet resistance),
- no or wrong arabinose (please make sure you use L-arabinose!)
- we observed that some strains (e.g. JM109, DH5alpha) are less efficient in RedET Recombination than others. DH10B, HS996, GeneHogs or TOP10 are our preferred strains.
- in very rare cases we observed that an elongation of the reaction time for the recombination from 70 minutes (incubation of electroporation) up to four hours was necessary for successful recombination.
- cells are not competent enough to take the linear DNA fragment up in the cells. Please make sure that the cells were kept on ice and that the water (respectively 10% glycerol) is sufficiently cold. Linear DNA has been shown to be about 104-fold less active than some DNA transformed in circular form (Eppendorf Operation Manual Electroporator 2510 version 1.0). Make sure that the time constant is around 5ms!
- please make sure that there was no arching during the electroporation process.
- please make sure that after electroporation the cells were plated on the appropriate concentration of antibiotics depending on the copy number of the plasmid or BAC.
Observation: Similar number of colonies on both plates, the induced and the un-induced one
If you obtain a high number of colonies on both plates, it indicates that there are still traces of the circular (or supercoiled) plasmid used to prepare the linear fragment left in the sample. Since the transformation efficiency of linear fragments is 104-fold less than of circular DNA molecules you may obtain a background even if no traces were visible on an agarose gel.
If the linear DNA fragment was obtained by restriction digestion, use less DNA and gel purify the fragment! If the linear cassette was obtained by PCR, set up a DpnI digest for your PCR product and gel purify it at the end!
If you obtain a very low number of colonies on both plates, it indicates that the overall efficiency of Red/ET Recombination is very low. In this case please control all parameters mentioned in the section for no colonies after Red/ET Recombination.
Observation:You can not separate the recombined plasmid after recombination from the non-recombined one even after re-transformation (high copy plasmid!)
In very rare cases we observed that after recombination it is difficult to separate the original plasmid from the recombined one. If you cannot separate them by dilution of the plasmid and re-transformation, you can choose a single cutting restriction enzyme and digest the plasmid. After re-ligation and re-transformation the two plasmids should be separated even when they were tangled before.
Question: Why is it important to shift the cells from 30°C to 37°C after induction with arabinose?
It is important that cells are incubated at 37°C, the temperature at which all proteins necessary for the subsequent recombination are expressed. There are about 5 copies of this temperature-sensitive plasmid per cell, and during one hour there is approximately 1 doubling step, meaning any daughter cell will still have on average 2-3 copies left and will also go on expressing the recombination proteins. The plasmid is actually lost after electroporation and recombination, when cells are incubated at 37°C overnight.
Question: Is it better to just linearize a targeting vector or cut it out of the backbone?
In fact, targeting vectors are usually just linearized rather than cut out of the vector backbone. The reasoning is that the vector will be eliminated at the recombination step. If the vector remains, it has not been a homologous recombination. We have not heard of situations where the vector would then reintegrate elsewhere. Cutting the backbone out would require an additional step of purification of the insert, which would be difficult and low yield because of its size.