- Q1: Uninduced cells (grown at 32°C) look "funny" or "fluffy" after electroporation and the 1 hour outgrowth.
A: First of all this is probably somehow λ-related (we don't see it in DH10B, the parent strain). It's perfectly normal and won't influence the outcome of your experiment. We don't see that with cells that have been heat-shocked. For some reason the 42°C heat-shock make the cells more homogenous (their cell cycle is synchronized for instance) and they don't show this "fluffy" feature. If you can't pipet around it, or if you plan to spread everything on a plate, you can pipet up and down a few times to "homogenize" the culture, before you briefly spin down the bacteria and remove the supernatant. This way you won't lose everything when you try to remove the supernatant.
- Q2: My EL350, EL250, and DY380 cells don't grow!
A:
(1) If you incubate these bacteria at 37°C they will die. These bacteria need to be
kept at 32°C. At this temperature the cI857 repressor is actively preventing
transcription of the recombineering genes (and the other genes that are part of the defective prophage present in these strains, especially the kil and gam genes - which, when expressed for longer than 15 min. will kill the bacteria).
(2) Only DY380 is tet resistant, EL250 and EL350 are not!
- Q3: Is the amount of salt (NaCl) in LB media important?
A: We use a "low salt" LB: 5 g yeast extract, 10 g tryptone, and 5 g NaCl for 1 L
media. Some recipes use 10 g NaCl. It seems that low salt works better for competent cells - not sure why - maybe there's too much residual salt left when the cells are ready for electroporation.
- Q4: Are EL250 and EL350 tet resistant?
A: No - in the process of making these strains, the tet resistance gene was removed
by homologous recombination.
- Q5: Can electrocompetent cells be stored in the freezer?
A: For transformations, DY380, EL250, and EL350 cells made competent by washes
in 10% glycerol can be stored, say, 6 months at -80°C. You can also perform the
heat-shock followed by washing in 10% glycerol and store these cells at -80°C.
However, the efficiency of recombineering will go down - maybe 10 fold. In
general we try to use freshly induced cells for recombineering, but frozen cells for
transformation (electroporation of plasmids and BACs). For EL250 and EL350 it
can be a good idea to make arabinose-induced and electrocompetent cells and
store these in the freezer for later use. This way you need only to do a transformation of a plasmid to get recombination between loxP (EL350), or FRT (EL250) sites.
- Q6: What are your conditions for electroporation?
A: 0.1 cm cuvette: 1.75 kV, 25 μF, 200 ohms; 0.2 cm cuvette: 2.5 kV, 25 μF, 200 ohms. The time constants should be around 4 ms (we see between 3.6 - 4.6). We use an electroporator from BioRad, and there may be brand-to-brand variation.
- Q7: Why do I get so many colonies on my un-induced control plate?
A: This high background is probably do to residual template (supercoiled plasmid template). If you use PCR: DpnI digest + gel purification. If you prepare your cassette by linerization, use less DNA (0.5-1 μg) and gel purify slowly - for example 25 V overnight.
- Q8: My BAC transformation didn't work!
A:
(1) Did you incubate at 37 °C instead of 32°C? If you did, then you killed your
bacteria!
(2) Another problem could be old BAC DNA undergone repeated
freeze-thaw cycles. Prepare fresh BAC DNA and try again.
(3) Try more
template, (up to 1 ug); you will have a smaller number of copies of a BAC compared to a similar amount of plasmid DNA. For example: 1 μg BAC DNA of 200 kb has the same number of copies as 20 ng of a 4 kb plasmid. (4) Maybe your competent cells are not good enough - should be 108 - 109 colonies per 1 μg supercoiled plasmid DNA. Test them with a supercoiled plasmid of known concentration, e.g. use 1 ng plasmid, make serial dilutions, and count colonies the next day.
- Q9: My BAC targeting experiment didn't work!
A:
(1) Make sure you have the right BAC. This is really important.You should single clone streak on selective medium, make minipreps followed by characterization by PCR and restriction digest before you proceed. Don't assume that the clone you ordered is what you think - it could be a mixture of more than one BAC - or just the wrong BAC!
(2) Did you make a control experiment with a non-induced control? If there's high background on the uninduced plate, prepare the DNA for electroporation better - DpnI digest and gel purify. Use less template for linearization (max 0.5 μg) to avoid uncut plasmid.
(3) Make sure you make good electrocompetent cells (108 - 109; colonies pr. 1 μg supercoiled plasmid is realistic).
- Q10: Do I need to select for tet resistance when using DY380?
A: No, it's enough to select for the marker present on the plasmid/BAC in the
bacteria. If growing DY380 up for preparation of competent cells (no vector), tet
selection might be a good idea. Remember that EL350 and EL250 are not tet
resistant.
- Q11: Do I need to select for sucrose resistance when using EL350 and EL250?
A: No.
- Q12: Do I really have to gel-purify my cassettes for electroporation
A: You should ALWAYS gel-purify you retrieval vector and targeting cassettes! Even if you do PCR followed by DpnI digestion - a tiny bit of residual supercoiled plasmid is enough to mess up the experiment. It's best to run the gel slowly overnight (e.g. 25 V) to ensure good separation
- Q13: How do I DpnI digest my PCR reaction?
A: Add 1-2 μl directly to the PCR reaction (works well in PCR buffer) after it's cooled down, and incubate for 1 hour at 37°C, followed by gel-purification. Use as little template as possible for the PCR reaction - 1-2 ng plasmid, and 30-35 cycles. This way the risk of plasmid contamination is minimal. If you use the same template over and over again, you could use a little bit of purified PCR-band
as template instead - this way you'll have no problem with plasmid
contamination. You should still gel-purify, however, to avoid other contaminations.
- Q14: Is the time constant after electroporation important for the succes of the
experiment?
A: Very much! With our settings (0.1 cm cuvette, 1.75 kV, 25 μF, 200 ohms) we usually get a time constant around 4 ms (3.6- 4.6). With a low time constant, e.g. 2.5 ms, the discharge happen too quickly, and the bacteria are killed instead of being porated. This is probably caused by to little resistance in the media - for instance if residual salt is present. Residual salt could have come from the DNA (you should always dissolve the DNA is ddH2O) or from the washing steps in making the bacteria competent.
- Q15: There seems to be an error in Liu et al. (Genome Res. 2003 Mar;13(3):476-84).
The order of the enzymes used for insertion of homology arms in PL451 and PL452 doesn't correspond with the enzyme order on the plasmid maps?
A: Actually, it's not an error; in the paper by Liu et al., the plasmid designations "PL451" and "PL452" are not used. The order of the recognition sites for the enzymes NotI, EcoRI, BamHI, and SalI does not correspond to the order of these enzymes in the plasmids you received. In short: Trust the plasmid map.
- Q16: The bacteria actually grew at 37 degrees?
A: When the bacteria strains used for recombineering are grown at 37 degrees, you select for point mutations that inactivate the prophage.
So, any surviving colonies after a 37 degree incubation will be
useless for recombineering
- Q17: Can you recommend a good kit for BAC purification?
A: We have had good succes with the Nucleobond BAC 100 kit from Clontech (BD Biosciences, catalog number 740579). It's a bit expensive, but it works well. Be sure to use the folded filter option in the manual. From 100 ml overnight culture expect 20-50 micrograms very good BAC DNA.
- Q18: Where can I get DOG (2-deoxy-D-galactose)?
A: Well, Ferro-Pfanstiehl has decided to discontinue this product. We recommend you try Sigma-Aldrich instead (catalog number D4407). There are probably other sources as well.
- Q19: Can I buy minimal media ready-made somewhere?
A: Yes, either from Amresco (catalog number E518), or from US Biological (catalog number M1015).
- Q20: What is the difference between DY380, EL250, EL350, and the new strains SW102, SW105, SW106?
A: The only difference is that the new strains can also be used for BAC modification using galK selection. In all other respects SW102=DY380, SW105=EL250, and SW106=EL350.
- Q21: Can I use the galK system for modifying plasmids as well as BACs?
A: The short answer is no. This system relies on the single copy nature of BACs, so counterselection will not work on multi-copy plasmids.
- Q22: Can I use recombineering for Cosmids?
A: We have not tried this, and the methods have been developed for BACs and plasmids. We expect you will get problems with cosmids, though, because of the lambda prophage.
- Q23: What kind of MacConkey agar do I need?
A: You need the Agar base, without sugar added, and then you add galactose. This is important, because you want to see if you have galactose-fermenting bacteria (gal positive), and if the MacConkey media has another carbon source already, then you get false positives.
- Q24: How long can I store the recombineering strains?
A: We recommend, upon receipt of the strains, that you make overnight cultures right away, and then make glycerol stocks for long-term storage. Use the glycerol stocks to start new cultures, there is no need to streak the bacteria on a plate first. The strains can survive up to one month at 4 degrees.
- Q25: Do I really need two shaking waterbaths?
A: Water is a much better conducter of heat than air, so the heat-shock works best in a shaking 42 degree waterbath. Furthermore, a shaking waterbath results in uniform growth, so we highly recommend it.
- Q26: How important is the OD600 for making competent cells?
A: You need to harvest the bacteria when they are in early-mid log phase, so the OD is important. We use an OD600 of 0.55-0.6. Please be aware that different spectrophotometers vary considerably, so you have to determine the optimal OD for your own spec.
- Q27: Can I use the minilambda system with the galK system?
A: No. To use the galK system you need to transfer the BAC to one of the strains SW102, SW105, or SW106. The minilambda system, however, can be used to make conditional knock-out constructs without the need to remove the BAC from it's original DH10B source.
- Q28: Can I use recombineering to "subclone" a large piece of DNA directly from genomic DNA?
A: Currently this is not possible. Part of the explanation is that recombineering (homologous recombination) happens during the replication cycle.
- Q29: Is longer homology arms better than short arms?
A: We find that inserting longer DNA fragments requires longer homology arms. Inserting up to 1 kb is normally not a problem with 50 bp arms, but to insert 2-4 kb fragments, you should consider longer arms, like 200-500 bp. For longer DNA fragments, you might need even longer arms. Of course, efficiency depends of the nature of the DNA, and in some cases short arms will work efficiently with even long DNA fragments.
- Q30: I cannot make electrocompetent cells, and I cannot get the pellet into solution when preparing the cells-am I using the wrong Falcon tubes?
A: You are probably using the wrong tubes-we use ROUND-BOTTOM 14 ml polypropylene Falcon tubes (cat. number 35-2059, Becton-Dickinson) for making competent cells. If you use CONICAL tubes you will have a hard time getting the pellet into solution.
