I am trying to transform recombinant plasmid to DH5α but I am not successful in doing it.
Here's the background information:
1. After gel purification of the restriction enzyme digested insert and plasmid: conc. of the insert: 58 ng/ul & plasmid: 19.1 ng/ul.
2. Ligation mixture (10ul):
ddH20- 5.17 ul
10X T4 buffer- 1 ul
plasmid- 2.6 ul (50ng)
insert- 0.73 ul (42.6ng)
T4 ligase enzyme- 0.5 ul
* length of plasmid is 8.8 kb & insert length is 0.75 kb.
Then overnight (18hrs) incubation at 16 degree cel.
3. Transformation:
10 ul ligation mixture to DH5α cells (100 ul) and mixing gently> placed on ice for 15 min>Heat shock at 42 degree cel for 45 sec>Ice for 3 min>1 ml LB broth to the mixture (DH5α-ligation mixture)> incubation (Shaking) at 37 degree cel. for 20 minutes> Centrifugation 12000 RPM for 2 minutes (making it 2x conc)>plating to agar plate (ampicillin).
4. No visible colonies.
So, I am asking for advice from experts. Thank you.
Follow these steps:
1. Pre-warm the solid LB dishes at 37 C.
2. Thaw the DH5-alpha bacteria on ice.
3. Add 3 μl of ligation mix into E.coli (maintain cold chain).
4. Incubate E.coli with ligation mix in ice for 15-20 minutes (gently tap the mixture occasionally to mix but DO NOT mix by pipetting). Turn on incubator and set it at 42C for next step.
5. Incubate briefly (just 20 seconds) at 42C and then return to ice.
6. After transfection of E.coli by heat-shock treatment, incubate it with 500 μl of SOC media (prewarm it at 42C) for 30 min in the shaker incubator at 200/min speed.
Next follow these steps:
1. After half-an-hour incubation with SOC, get the transformed E.coli, pre-warmed agar plates, inoculator, plate rotator, and flame on the bench top.
2. Label two agar plates – 100μl and 400μl. Take another plate as (+) ve control and label accordingly.
3. Take 100μl of the cell suspension in the 100μl labelled plate and streak with the help of plate rotator until it dries.
4. Centrifuge the remaining cell suspension at 6000 rpm for 1 min and discard 300μl supernatant from top.
5. Streak the 2nd plate (400μl labelled) with the remaining concentrated cells (first mix gently with pipetting as the competent cells are very weak) and keep on streaking with the plate rotator until the surface dries up.
6. Incubate all plates in the incubator at 37C for 12-16 hours.
Hope this helps!
Dear A.T.M
There are many reasons because a ligase cannot provide colonies.
The most obvious are:
1) Low efficiency competent cells (if you are using hand made competent cells)v
2) No good digestions of the inserts or of the vectors?
3) Error in the antibiotic selection
Moreover i see some details in you protocoll that can be improved from my poin of view:
I think that you have to extend a little the timelines of some steps to improve trasfromation efficiency:
eg: 30' instead 15' in ice
60' instead 20 minutes shaking at 37°C after 42°C shock
and moreover i think that is preferable avoid the centrifugation step (expecially at so high speed) but trasform lower amount of bacteria (eg 2ul of ligase in 25ul cells), resuspend it with 250ul of LB and plate all.
However for the future i suggest to you to evaluate enzime free cloning approaches as PIPE cloning or In-fusion cloning.
On my blog ProteoCool ( https://proteocool.blogspot.com/ ) you can find several information about PIPE cloning.
good luck
Manuele
How were the chemically competent cells prepared? Did you use the CaCl2 method? Using RbCl2 instead could increase the efficiency of the competent cells quite a bit, so try preparing the DH5s that way.
May I also ask how old your competent cells are? Chemically competent cells can lose efficiency while frozen back, especially if they are old. And with ligation mixtures you are going to want high transformation efficiency. I would try making a fresh batch of competent cells if they are over 6 months old.
Lots of other good answers for trying to improve cell recovery after heat shock, is- pre-warming media and plates, longer outgrowth step, and using SOC medium for outgrowth rather than LB. If you decide to outgrow for 60 minutes, ditch the centrifuge step. Also, 12000rpm on what rotor? What are the actual g forces produced? If you don’t want to hurt the cells you can pellet them at 5000xg for 3 minutes and that will be more gentle. the cells are still likely pretty stressed from the heat shock at this point so I personally wouldn’t centrifuge to concentrate at all.
You should also add a viability control after transformation, where you plate both an un-transformed control and the transformed cells on both antibiotic containing and antibiotic free plates. This will tell you if something you are doing during the procedure is killing your cells, or if your transformation efficiency is just low.
Lastly, if you haven’t checked already, make sure that the ampR gene on the vector is not deficient. Transform some cells with the empty plasmid and see if you have a similar issue. If the empty plasmid works fine, then something about your restriction digest/ligation reaction did not work out.
hope this helps!
You need to do the proper controls to determine whether the problem is with your transformation or competent cells, is with your DNA, or is with the ligation protocol. As Arlind Mara mentions, be sure to do a control with uncut plasmid (preferably the uncut plasmid you are using for your experiment) and you should get hundreds of thousands of colonies on your amp plate if everything is good. If that works well then methodically check the other steps. Is your DNA gel purified and if so are you sure you have intact DNA? Are you sure your ligase and buffer are working well.
just check the antibiotic concentration on your agar plate ,this happened with me many times with amp ,try to decrease the concentration of amp little bit as the amp is bactericidal not bacteriostatic and in many cases can kill the bacteria cells ,also try to check the insert concentration before ligation and increase the ratio of insert to vector to 12:1 if the concentration of insert is low ,i hope this help
Thank you very much for your kind suggestions. Now it's working well. Muhammad Aizaz Amy Johnson
Mohamed Halawa Michael J. Benedik Arlind Mara Manuele Martinelli Zahir Hussain
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