Total absence of cells after transformation:

- As you mentioned already heat inactivate restriction enzymes, then as suggested above gel purify both the insert and vector after digestion

- depending on the amount of DNA and restriction enzyme used 2 hours might be not sufficient. Using too much restriction enzyme can also cause inhibition due to the glycerol in the buffer

- In your experimental setup it is difficult to verify if restriction did work properly (e.g. by the ligation controls mentioned below), since only small DNA fragements are removed at the ends of the insert and within pET28a --> blunt-end subcloning of the uncut insert DNA fragment can help sometimes because you can verify if the fragment has been cut properly with NdeI & XhoI by gel electrophoresis, which is not the case if you just add short overhangs. Similarily, using a pET28a derivative with insert would help to analyze if pET28a was cut properly.

- add extra ATP to the ligation reaction (ATP in the ligation buffer can degrade due to repeated freeze-thaw cycles)

- ligation: 16°C over night or several hours at room temperature

- optimize ligation volume & DNA concentration

- dilute ligation before adding to competent cells to ensure that DNA and cells get mixed properly

- Check your transformation efficiency - should be in the range of 10^6 - 10^8 cfu/µg DNA for self-made chemically-competent cells and pUC19 plasmid

- optimize the heat-shock transformation protocol: e.g., growth of cells in the absence of antibiotics after heat-shock depends on the resistence marker used (at least 45 min for ampicillin or 1h 15 min for kanamycin), prewarm plates and media, ...

Possible solutions if you have to many "wrong" colonies on your plates:

I would suggest to include ligation controls, i.e., treat your NdeI & XhoI cut pET28a plasmid with ligase (without adding insert DNA) and transform it into DH5Alpha. Similarily, transform NdeI & XhoI treated pET28a without ligation. Based on these controls and the amount of colonies you obtain, it is possible to judge if the plasmid preparation contains uncut plasmid, and plasmid which was only cut with one of the two enzymes.

Furthermore, it is possible to minimize background (i.e., colonies that contain pET28a without insert) by treating the ligation reaction that contains vector & insert with a restriction enzyme that cleaves at a site located between the Nde & XhoI site , e.g., HindIII or BamHI (Important: This site should NOT be present in your insert) to digest uncut or single cut and religated pET28a (if present within the preparation).

when you introduced the RE cut sites in the pcr primers did you add and extra 3 bases at the 5' end of the primers so that the RE could bind to the pcr product prior to cutting?

Paul Rutland Yes we introduced roughly 6 dummy base pairs next to our restriction site.

you need to define what you mean by not working. Are you getting many cells that are not correct? then the problem is that you did not gel purify your plasmid which causes a small background of undigested plasmid to transform the cells. Are you not getting cells at all? check the ligation reaction by amplifying using primers that amplify in the plasmid and the insert preferably use two primers that check across the ligation junctions if you can. If you get a positive band from this then do an ethanol precipitation of your ligation reaction and retransform. If the reaction is negative repeat the ligation with 1:10 ratio and check and transform again.

Hanna Alalam Thank you Hanna! Sorry for me being unclear. With not working I meant a total absence off cells after transformation (except for the positive control). I will try your suggestions!

Total absence of cells after transformation:

- As you mentioned already heat inactivate restriction enzymes, then as suggested above gel purify both the insert and vector after digestion

- depending on the amount of DNA and restriction enzyme used 2 hours might be not sufficient. Using too much restriction enzyme can also cause inhibition due to the glycerol in the buffer

- In your experimental setup it is difficult to verify if restriction did work properly (e.g. by the ligation controls mentioned below), since only small DNA fragements are removed at the ends of the insert and within pET28a --> blunt-end subcloning of the uncut insert DNA fragment can help sometimes because you can verify if the fragment has been cut properly with NdeI & XhoI by gel electrophoresis, which is not the case if you just add short overhangs. Similarily, using a pET28a derivative with insert would help to analyze if pET28a was cut properly.

- add extra ATP to the ligation reaction (ATP in the ligation buffer can degrade due to repeated freeze-thaw cycles)

- ligation: 16°C over night or several hours at room temperature

- optimize ligation volume & DNA concentration

- dilute ligation before adding to competent cells to ensure that DNA and cells get mixed properly

- Check your transformation efficiency - should be in the range of 10^6 - 10^8 cfu/µg DNA for self-made chemically-competent cells and pUC19 plasmid

- optimize the heat-shock transformation protocol: e.g., growth of cells in the absence of antibiotics after heat-shock depends on the resistence marker used (at least 45 min for ampicillin or 1h 15 min for kanamycin), prewarm plates and media, ...

Possible solutions if you have to many "wrong" colonies on your plates:

I would suggest to include ligation controls, i.e., treat your NdeI & XhoI cut pET28a plasmid with ligase (without adding insert DNA) and transform it into DH5Alpha. Similarily, transform NdeI & XhoI treated pET28a without ligation. Based on these controls and the amount of colonies you obtain, it is possible to judge if the plasmid preparation contains uncut plasmid, and plasmid which was only cut with one of the two enzymes.

Furthermore, it is possible to minimize background (i.e., colonies that contain pET28a without insert) by treating the ligation reaction that contains vector & insert with a restriction enzyme that cleaves at a site located between the Nde & XhoI site , e.g., HindIII or BamHI (Important: This site should NOT be present in your insert) to digest uncut or single cut and religated pET28a (if present within the preparation).

Here is the protocol we follow in our lab:

Insert Preparation

1. Gel purify the PCR product

2. Double digest PCR product for 1 h at 37 degC using -HF enzymes in Cutsmart buffer from NEB.

3. Purify PCR product again using PCR purification kit.

Vector preparation

1. Double digest vector using the same set of REs and buffer. You might add alkaline phosphatase(e.g. FastAP) at this step to prevent the self-ligation of vector (optional for double digestion)

2. Gel purify the vector

Ligation

1. combine vector and insert at a molar ratio of 1:3-1:5. You can do the calculation here https://nebiocalculator.neb.com/#!/ligation

A typical ligation mixture is given below:

vector - equivalent to 50 ng

insert- X uL (need to calculate)

T4 ligase - 1 µL

10X ligation buffer - 2 µL

ddH2O - upto 20 uL

2. incubate at RT for 1 h

3. transform 2 uL ligation mixture into 50 µL of competent cells

Notes:

1. make sure REs don't cut within insert

2. add 3-4 bases upstream of restriction site in primer

3.make sure both enzymes are optimally active in the buffer system used

4. thaw the ligase buffer at room temperature and dissolve the DTT completely

5. remember vector:insert ratio is a Molar ratio!

5. don't heat inactivate the ligation reaction

6. don't transform too much ligation reaction. It should be less than 10% of the final volume of transformation reaction.

7. always include a negative control(vector only)