I have been trying to do the transformation for the past couple months, but I have no luck on getting the high efficiency.
I have tried to make the inserts, digested them with sacI and speI restriction enzymes at the same time, did gel purification to purify the insert dna, and run on the gel again to compare if I got the insert digested properly.
I did the same procedures with the vectors, except digesting them sequentially with sacI first, and then speI.
I tried to treat vector with or without phosphatase,and treat kinase with or without kinase, and ligate the vector and insert with 1:5 and 1:10 ratio with two different ligate from either promega or thermo science.
The ligation condition was either at RT for 2 hours, or at 4 degree C for overnight. And then, I transformed 100 ng of the ligated product into 100 uL of comp. cell with 9x10^8 transformation efficiency.
However, the average transformation efficiency for the ligated product was only around 10^4. The one for ligated product with treatment of phosphatase and kinase was even worse than the one without treatment.
I still got around 10% of wildtype for my colony pcr and 10% unsuccessful ligated products.
Please help me... any commons and suggestions will be appreciated.
It is not entirely clear what your question is. In any case, take into account that transformation efficiency figures are usually estimated with supercoiled, intact plasmid DNA. The efficiency of transformation of ligation reactions is usually much lower (although I do agree that a 5-orders-of-magnitude difference is too large).
It is best to assess the efficiency of the cells under your own conditions, using 1 uL of a control preparation of pUC19 or pBR233 at 1 ng/uL. Perhaps there has been a drop due to improper storage during shipping, and in any case, a control will tell you what efficiency to realistically expect and whether to take matters to the technical support department of your supplier.
Transformation efficiency depends on vector-insert size and bacterial strain you use. Positive clone selection is important too. I usually achieve a good efficiency when I am looking for
The efficiency of ligation depends in many cases on the vector you use and the length of the fragment. For inserts < 3000 bp I usually amplify them by PCR using Pfu Polymerase with primers tailing the restriction site and 2-3 additional bp due to digest the PCR products directly. So what I usually do is:
- I amplify the gene of interest (2 PCR reaction of 50 ul)
-purification of PCR products
- digestion of the insert and vector with restriction enzymes
-purification of the products (vector from the gel and the insert directly from enzymatic reaction)
-quantification and reaction of ligase
For ligase reaction I usually use no more than 50 ng of the vector and the maximum volume of insert I can put (very often the ratio is more than 1:10) and usually it works quite good;)
I hope it was helpful
Thank you all for answering my question. To Alejandro Martin, I had use pUC19 as the positive control, and the transformation efficiency of the competent cell was good for sure.
To Ignacio Montero, the size of insert was only 85 bps, and the vector was around 4000 bps. I believe the size of the ligated product should not be an effect. I also tried to use chemical competent cell to transform the ligated DNA, but the TE was even worst than the one transformed via electroporation.
To Anna Maciag , I will try to ligate with more insert and see if this helps.
Thank you so much again for all of your helps. I really appreciate it.
Oh, so you're using electroporation! Are you inactivating the ligase?
- Badges
- Science topic