I did a bacteria transformation and ended up with low transformation efficiency for all my plates. Here's the protocol I used following addition of competent cells to the plasmids and mixing, and before adding it to my plate.
1. incubate on ice for 10 min
2. heat shock in 42˚C water bath for exactly 2 min.
3. incubate on ice for 2 min
4. add LB w/out antibiotic
5. incubate in 37˚C shaker for 30min
I followed every step as stated but did have to wait for 90min between steps 4 and 5 (w/ tubes on ice) due to technical issues with the shakers. Assuming that my cells are of a reasonable competence and none of the other steps went wrong, could the delay in this single step have caused the low TE? And what's the purpose of the shaking step except for an even distribution of nutrients?
It is unlikely that putting your cells on ice would dramatically reduce transformation. The purpose of the shaking step is to ensure good aeration of the culture, otherwise the cells will go partially or completely anaerobic.
The protocol you are following is a low efficiency protocol (it is just faster). Make the following adjustment:
1-incubate on ice when you add DNA for 30 min.
2-shock for 30-45sec.
3-recover shaking in LB with proper aeration for 1 hour (do not just add LB to the eppies) use 13 ml falcons.
I agree with Michael the additional ice incubation step is unlikely to have caused a big drop in efficiency (just for reference you can leave it at room temp if you run into the same problem again instead of incubating on ice).
I guess your big concern should with 2 minutes heat shock. I ready many protocols saying 30, 45, 50 or 1 minutes at more in heat block. I don't know if these increasing related to water bath usage or not.
for incubation time before spreeing on LB agar plate its depend on your antibiotic.
The delay should not have decreased transformation efficiency. The use of the 37C incubation is to allow the cells to begin growth and ensure that the antibiotic resistance casette on the plasmid has some time to be expressed before putting them on a plate with antibiotics. If you do not do this step, and you are using an antibiotic that blocks the ribosome from functioning, it would greatly decrase the cell's ability to grow (may get a super low TE). I would follow Hanna's protocol as mentioned above, but have never seen a need to incubate in a falcon, 1 ml of culture in a 1.5ml epindorf tube has always been sufficent.
Hi Luna:
I routinely do transfections & get fairly high transfection efficiencies. You may try the following if you wish :
1. (50ul competent cells + 1-2 ul DNA) --> incubate on ice for 30 min (pre-warm your SOC medium, 37C).
2. heat shock in 42˚C water bath for 40 sec.
3. incubate on ice for 2 min (place antibiotic plates in incubator to pre-warm)
4. add pre-warmed, 37C, SOC to your transfection (rich medium, aids cell recovery post-heat-shock).
5. incubate in 37˚C shaker for 1 hr.
6. Plate on pre-warmed antibiotic plates & get a good night's sleep.
Good Luck,
Ron
Hi, Luna Vanhorn
As per your question, I would suggest the following things,
Incubate your competent cells on the ice up to 30 min with your Ligated product/ DNA.
Heat shock for 50-60**sec.
add LB after 2-3 min incubation, further incubate at 37°C* for 2 h.
after that, you can centrifuge your competent cells and add 100 or 200 ul LB and plate on your respective plates.
* 37°C/200rpm is the best temp. for good growth/aeration for E.coli.
** if you are giving heat shock in a water bath it should not be exceeding 50-60 sec.
if you are giving in the heating block you can go up to 90sec.
I don't know if it is the procedure that asked you to heat shock at 42 deg for 2min. Normally heat shock at 42 deg for 30 sec is OK. Probably, extending the time to 2mins may likely affect the transformation efficiency.
is this the vector created via REs and ligation? or using a pcr/gibbson method?
The incubation at 37C as stated is to let the bacteria recover from heat shock and allow the production of antibiotic resistance gene (Not usually necessary for Amp). We use SOC media (Suppressor of catabolism) media not LB at 37C because the point here is not to get bacteria to grow but to recover and produce resistance. 2 minutes on ice is necessary to allow the bacteria to cool down/recover after heat shock: 2 minutes is ok, longer is also ok. There are of course many ways to do the same thing.
As noted by others, the key step in transformation is the heat shock -- the temperature and the duration; 2 minuts is probably too long. Note that the type of the vessel you use at this step and its heat exchange properties may also affect the results.
Sorry for not getting back to you till now and thank you so much everyone for all your responses; they have been plenty helpful. Having read all your inputs and discussed it with my colleagues I agree that the heat shock step might be the problem, and I also agree with Hanna Alalam that this protocol can indeed use some improvement on the TE. We will modify the heat shock temperature & time and hopefully see some improvements. Aleksey Karpitskiy the vectors were made by REs and ligation.
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