Hi Sandeep,

>It is basically concentrations problem in your reaction set up I feel. As Abeeb Abiodun Yekeen mentioned your are using too much of plasmid, just go with 1 ug each of plasmid as well as insert concentration and find out concentrations of digested products prior to ligation set up.

> I agree with Sebastian Schmitt

> Here is the brief protocol. Set up a 20uL double digestion reaction followed by 15uL ligation reaction and use 5-8uL for transformation.

Digestion: 1ug of DNA+2uL of Cut smart buffer (10x if your using NEB product)+1uL of each enzyme (according to your mentioned stock conc.)+ rest make up with sterile milliQ. Incubate at 37oC for 3 hrs, estimate the digested products concentration.

Ligation: ~200ng of digested vector you can use for ligation and calculate the required insert conc/volume accordingly. Incubate at 16oC for O/N

Good luck

Hi...

With regards to the insert, 3 bases upstream of the restriction site is sufficient for both NdeI and XhoI if you are using Thermo's fastdigest enzymes.

As for the vector, you might want to reduce the concentration of the plasmid being digested. The lower the amount of plasmid in the reaction mixture, the higher the chances of having a COMPLETE double digestion of the plasmid; this is more so considering the closeness of the two sites on the vector... 1 hr is more than sufficient for FD enzymes, but you can consider extending it to perhaps 2 hrs to rule out the decrease in the efficiency of the enzyme with time.

However, you might want to consider choosing a more distant site from the first one (perhaps BamHI) if you still can't get the ligation to work.

Hi...

If you want to confirm your insert, then just isolate plasmid and run pcr with gene specific primers.

agree with Sebastian Schmitt

regards

Hi

for double digestion of the vector try each enzyme separately and in both order (xho then nhe and nhe then xho) with a column purification in between and at the end and then do two ligations (sometime one digestion order is better than the other...). You can also try a 3 ways ligation by cutting the vector with pst1+nhe1 and (in another tube) pst1+xho (pst is in the amp gene (or pvu1)); purify the right fragments (small pst-xho and large pst-nhe) and ligate with your insert. To digest pcr fragment your primers should have several extra bases (do not hesitate to add 6 or 8 more base at the 5' ends even if books say that 3 is enough!!) and put a lot of enzyme (10u) or purify the pcr fragment before digestion (the pcr buffer is not efficient for all restriction enzymes) and then after digestion too. should work

you can:

1. change your buffer.

2. change your ligation condition.

3. run your ligation product on gel and do gel extraction from the fragment which is your thing.

Hi Sandeep,

>It is basically concentrations problem in your reaction set up I feel. As Abeeb Abiodun Yekeen mentioned your are using too much of plasmid, just go with 1 ug each of plasmid as well as insert concentration and find out concentrations of digested products prior to ligation set up.

> I agree with Sebastian Schmitt

> Here is the brief protocol. Set up a 20uL double digestion reaction followed by 15uL ligation reaction and use 5-8uL for transformation.

Digestion: 1ug of DNA+2uL of Cut smart buffer (10x if your using NEB product)+1uL of each enzyme (according to your mentioned stock conc.)+ rest make up with sterile milliQ. Incubate at 37oC for 3 hrs, estimate the digested products concentration.

Ligation: ~200ng of digested vector you can use for ligation and calculate the required insert conc/volume accordingly. Incubate at 16oC for O/N

Good luck

Agar gel electrophoresis

Dear, I think you can try reducing the DNA concentration or increasing the enzyme concentration, increasing incubation time. But if the restriction sites are separated by just 3 bases, it might be hard for the enzymes to be efficient.

Good luck

Hi Sandee,

do you add the phosphatase to your plasmid to avoid the self ligation. and this step is before the ligation, adding the phosphatase to your digestion reaction just for vector for one to two hours and then run both reaction ( inset and vector) and purify the the right DNA by gel purification Kit and set the ligation reaction dependent on the DNA concentration. its prefer the ratio 3:1 Insertion: ligation.

and think your digestion time was not enough.

good luck

For checking double digestion of insert, adds an extra step to your cloning procedure wherein you need to clone your PCR product (blunt ended) in a plasmid which will be blunt end ligation. So, basically you can do this in any of the plasmids by using a blunt end cutter RE. From this clone you can excise your PCR product with NdeI and XhoI, the insert release can be gel purified. This will assure the double digestion of your gene of interest.

Or else optimise your double digestion reaction, keep for longer incubation (overnight) in 100 uL reaction volume (as DNA is huge). It should work.

Dear Sandeep,

The sites for XhoI and NdeI are indeed very close to each other (7bp). The mechanism of restriction is such that the enzyme must wrap around at least one turn of double helix (10-11 bp depending on conformation). Also, many restriction enzymes have long residence times even after cutting and can prevent a second enzyme from accessing the cut site. Therefore you need to carry out the reaction in steps:

1. Digest with NdeI first (this has greater problems cleaving close to the end compared to XhoI).

2. Gel purify the linear band, then cut with XhoI.

3. After the XhoI cut, treat the DNA with phosphatase to prevent self ligation of any single cut DNA remaining in the sample

4. Gel purify and use for ligation to insert.

Notes:

Since there are numerous manipulations you need to start with higher amount of DNA therefore the digestions need to be carried out in larger volumes (100 uL per 10 ug).

Remember that linear DNA migrates more slowly than supercoiled but faster than nicked when you are cutting bands out of gels.

Use 0.7% gel and run for longer to get good separation of the linear band.

Good luck!

Deepan

Hi, Sandeep Kumar

The best option and the protocol that I used, is the one proposed for Eswar Reddy Maddi in the answers:

Digestion: 1ug of DNA+2uL of Cut smart buffer (10x if your using NEB product)+1uL of each enzyme (according to your mentioned stock conc.)+ rest make up with sterile milliQ. Incubate at 37oC for 3 hrs, estimate the digested products concentration. Verify that both enzymes using the same reaction buffer.

Ligation: ~200ng of digested vector you can use for ligation and calculate the required insert conc/volume accordingly. Incubate at 16oC for O/N. Here, you can use the size of the insert and vector and calculate what is the best concentration of fragment that you can use. I worked with 1:3 and 1:5 concentratios, in other hands, for each ng of vector 3 or 5 ng of insert.

Finally, verify the fragments generated of the double digestion and I recomend use controls, single digestions only for verify that both enzymes whit concentration sugested, was cuting ok. The results generated,you can see in an agarose gel.

Hi,

Maybe you could try adding CIP at the end of the digestion, for about 30 minutes, this is going to prevent re-ligation.

4ug pladmis is too much specially if dissolved imn TE buffer

The best way of doing the ligation is to set up two digests in two tubes:

Digest 1: Cut vector with PstI and NdeI and isolate 4627 bp band from gel

Digest 2: Cut vector with PstI and XhoI and isolate 1074 bp band from gel

Use both isolated bands together with your insert in a 3-fragment ligation.

Forget about the other suggestions. And forget about phosphatases, they are not necessary here. If my procedure does not work there must be a problem with the insert. Think about cloning your PCR product into a plasmid.

Best wishes

Jürgen

You are getting transformants with empty vectors because not all plasmid molecules were cleaved with both enzymes. Even traces of uncleaved or single-site cleaved vector molecules can yield a very strong background of religated molecules that you observed.

My suggestion is to first add NdeI as it has less problems cleaving supercoiled DNA, then after 1 h add XhoI which is far more problematic with scDNA. Also, keep in mind that one enzyme unit does not mean the same for all enzymes and all plasmids. Based on unit definition, you would need much more XhoI (by volume), as there is only one cleavage site present on lambda phage, while NdeI has 7 sites (1 U cleaves 1 ug of lambda in 1 h by definition). So, if you need to start with 4 ug for whatever reason, increase especially the volume of XhoI (to e.g. 3 ul) and prolong the total reaction time to 3 h. Do not prolong the reaction time to overnight as it can result in removal of some additional bases from termini (4 h max. is recommended by NEB for NdeI).

i just want to add that there is a reason why you do not need phosphatases ( i have made this statement in my previous post).

if the vector is perfectly cut, it has two different ends that cannot religate without picking up insert. if not, you will have singly-cut vectors that can not take up insert. these can religate, showing up as colonies on the plates. a huge number of colonies in vector-only ligations makes you aware of the existence of singly cut vectors, which is good. .

If you add phosphatases, the singly-cut vectors cannot religate, the vector-only control ligation will not show many colonies. thus you lose the information on the quality of the double digestion.

another reason why phosphatases are completely unnecessary comes here: although they may reduce the number of colonies on your plates, their use will not create more vectors with two different ends that can pick up an insert. however, this is exactly what you need for a successful ligation. so try to increase the number of vectors that have two different ends! when the sites as close together (as is the case with you) the only save thing to do is t follow my instruction above. it is save because you can see on the gel if both enzymes have cut.

Best wishes

J