For the luciferase - make a new question and give more info on what cells, what constructs etc you are using; are you using positive controls (that may come with your setup), etc pp.

CLONING STUFF HERE:

I see nothing generally wrong in your cloning setup.

First thing you should check is the running buffer/gel buffer. If it´s TBE, that could be the problem. Switch to TAE if you really really really have to do gel purification.

One more thing that can cause trouble - and should be avoided if possible: Gel-purification. You don´t write if you are using "prep-light" and how long it takes you to cut your bands from the gel. Exposure time should be minimized. Load your gel in a way that you have enough empty wells between different products so you can cut slices for each product without UV light and then only put one slice on the UV table at a time to cut out the required band. IF your UV-table doesn´t come with prep-light function, try to avoid the gel-extraction step (see below).

Transformation: I think it´s fine, usually commercial compenetnt cells work very good. But you could of course transform one aliquot of cells with 1 uL of undigested plasmid to make sure that transformation and selection (correct antibody) isnt the problem. -> I usually plate 10 % of the bacterial solution on one plate, spin down the rest (10 000 rpm for 1 min), decant the supernatant and resuspend and spread the pellet in the 50-150 uL of flowback (the drop that doesnt fall off during decanting); so that way I am actually spreading everything and if the trafo worked well I can pick from the 10% plate, if not I have the 90% plate.

Ligation: you don´t write how much plamsid you use. Usually 25-50 ng is enough. 100 ng also works. But more isn´t always more, so best to stick to what your T4 kit manual says. (I use rapid ligase from Thermofisher, ligation time 20 min, works fine, transformation of 1-2 uL into TOP10 chemically competent bacteria)

Restriction digest: looks ok, and since you don´t report seeing uncut plasmids, I don´t suspect any problems here.

Some suggestions:

- I use rSAP from NEB, it can be used directly in the restriction reaction alongside the restriction enzymes and can be heat-inactivated.

- why do you gel-purify your plasmid backbone? You are de-phosphorylating it AND using two different enzymes. De-phosphorylation makes (re-)ligation impossible, so a quick DNA/PCR cleanup kit would be sufficient. If whatever you are cutting out from your plasmid is 1000 bp) you can use one primer in the insert and one in the plasmid. Colony PCR: pick a colony with a yellow tip, smear it into 100 uL ddH2O (in PCR-strips) and then make a backup of that colony on 1/8th of an agar plate with the required antibiotic(s) by making a small streak. Once you have picked 16-48 colonies in that way, heat the PCR strips with your colonies at 95°C for 5 min. Run your PCR, use 1 uL of the colony-lysate.

Dear Sokviseth, Dr. Christoph has answered in a very comprehensive answer above.

Why dont you shift to infusion or gateway cloning instead? So many factors are involved in our failure while performing conventional cloning; whearas other methods reduce this factors. I wish you good luck

My initial guesses are:

1. UV damage.

2. Chaotropic salt carry-over from the gel extraction. What do your 260/230 ratios look like afterward? A little is fine but too much can ruin your nanodrop readings and potentially denature the ligase.

3. Ligase buffer has gone bad. Usually you can give the thawed buffer a sniff test to get an estimation, the smell of DTT gets less intense the older the buffer is or the longer it's been left out at ambient temperatures.

You might want to try switching to doing gel purification with a blue-light excitable dye like SYBR safe and using a blue light transilluminator. That way you can exclude UV damage as a possibility, also at least for me being able to take my time cutting out the bands tends to result in cleaner, better yields because I don't have to dissolve a bunch of extraneous agarose.

Thank you so much Dr. Christoph Metzendorf for your comprehensive and useful answers. I really appreciate your time and kindness.

> For the plasmid amount I used in ligation is around 20 - 50 ng. So I think that this amount is not really a problem since you have also mentioned this range of amount of plasmid.

> Since I do not have UV table in my lab, I use only UV lamp to cut out the required band. Therefore I think that the possibility of DNA damage by direct exposure to UV is quite high. Plus the exposure of UV is long as well. Furthermore, I think it is quite hard to cut the band without UV exposure because I cannot see the band unless there is UV light.

> Moreover, I am not really sure about DNA/PCR cleanup kit since I have never used this method before. Would you mind clarify in detail the technique and purpose of this???

> For Colony PCR, since there is no colony appeared on the next day, I cannot do colony PCR to screen my colonies.

> One more thing, after doing gel extraction, I detected the low A260/A230 (0.02 - 0.8) in few samples with concencentration around 10 - 20 ng/uL. Is the low amount of A260/A230 ratio is because of contamination or the low concentration????

Thank you so much Dr. Allahbakhsh .. for your recommendation. I really appreciate your kindness and time.

However, I am not really sure about in fusion or gateaway cloning method. Would you mind sharing me the detailed protocol of this cloning technique???

Thank you so much Alexandra Johnson for your useful answers.

1. I believe so because I use UV lamp to cut out the required band, so that the direct exposure of UV on DNA band is quite high and long.

2. After doing gel extraction, I detected the low A260/A230 (0.02 - 0.8) in few samples with concencentration around 10 - 20 ng/uL. And the ratios of some samples are quite okay (around 1.7 - 2.0). Is it because of the presence of chaotropic salt or the low concentration of DNA that contributes to low A260/A230 ratio??? Moreover, do you have any techniques to remove the chaotropic salt???

3. I normally make aliquot around 10uL of ligase buffer and thaw it in room temperature around 2-3 hours to make sure there is no more precipitation present inside the buffer.

Furthermore, could you tell me the blue-light excitable dye you use in the gel purification???

Sv Mg

GEL purification: please try TAE buffer if you are using TBE!! This can solve your problem with little effort. Gel purification failed for us with samples cut from TBE gels; no problems with TAE + prep-UV (always enough clones and no mutations).

There are many suggestions now, I´d recommend doing some control things:

Check if your enzymes can be heat-inactivated. If so: digest your plasmid and PCR product as you did before (including the de-phosphorylation if the enzyme can be heat inactivated). Heat inactivate all enzymes.

A; from your plasmid take an aliquot of 20-50 ng and from the PCR product use an aliquot corresponding to 1:3 (plasmid:pcr product) molar ratio and use these for ligation. Transform 1-2 uL

B; do the gel purification with the lamp as far away from your gel as possible and use TAE for running the gel. Ligate as you usually do and transform 1-2 uL.

C; transform undigested plasmid (2 ng) into your bacteria.

-If all works, it might have been the TBE buffer.

-If only B fails: your UV lamp might really be too strong or your DNA way too dirty; precipitation with ethanol can do the trick here

-If only C works: you ligase might be the problem

-if nothing works: your transformation is the problem

Answers to your other questions.

DNA cleanup kit:

thats just a kit for buffer exchange and removal of proteins. Basically, you bind your DNA to a spin column, wash with 1 to 2 different buffers and elute

e.g. https://international.neb.com/products/t1030-monarch-pcr-dna-cleanup-kit-5-ug#Product%20Information

UV:

- if you use a UV lamp, it´s not necessarily stronger or more harmful than a non-prpUV table; it depends on the wavelengths it generates and also on the strength; WITH a lamp you you could reduce the amount of radiation by moving your gel as far away from the lamp as possible.

- BE QUICK! ;-)

- with leaving lanes free between different samples I meant that you can cut between those lanes, so you have one chunk of gel per fragment that you want to cut. While you cut fragemnt "A", the gel-chunk with fragment "B" would be not exposed to UV light. Also, as soon as you have cut your band, switch off the lamp.

- if you want to load more, to get a bit higher DNA concentrations, you can use normal clear sticky tape wrapped around two or three teeth of your comb for making the wells in your gel.

-260/230 readings can be a bit low, especially with gel extaction; and yes, they tend to be lower with lower DNA concentrations as the contamination with the respective compounds is a bit constant, so relatively higher if there is less DNA.

You didn´t write if you have single PCR products. The plasmid: you don´t need to do gel purification if you use dephosphorylation... .

ColonyPCR was a suggestion for a setting where you would not do gel-purification of a PCR reaction that does produce several different products. Instead of extracting plasmids from 100 colonies and doing restriction analysis, it´s more efficient to first go for colony-PCR to quickly identify potentially good clones, then extract those plasmids, do restriction digest analysis and sequence only 1-2 that have inserts of the right size, to make sure there are no mistakes in PCR.

Sv Mg

The low concentration of DNA contributes, but with the values you listed probably a lot of it is from salt contamination which is common in gel extractions. One way to lessen it is to leave the wash buffers on the column for a few minutes each time instead of immediately spinning, which gives salts more time to solubilize off the column. Another way to lessen it is to just use a newer kit (assuming you are using a kit), I've noticed with gel extraction kits that the buffer which dissolves the agarose starts off clear but goes yellow-orange over time (oxidation probably?) and usually the older the kit the worse 260/230 ratio I get at the end. I don't know if that means the colored contaminant in the buffer just absorbs UV very strongly or if it just isn't very soluble in alcohols and doesn't come off during the wash steps.

As for the SYBR safe stain: https://www.thermofisher.com/order/catalog/product/S33102#/S33102

It's pricier than ethidium but that isn't too much of an issue for me because I only use it for gel extractions so a single tube of it lasts a very long time. You need a transilluminator which can light up in a blue wave length instead of UV, and either an orange filter or orange glasses to block other emission wavelengths. The illumination happens within the visible spectrum, unlike with UV, so without a filter to block most of it you'll just end up staring at a blindingly bright blue light in a dark room and won't see anything. You can illuminate it with UV but obviously that defeats the purpose. I haven't this myself to know if it actually works but you might be able to cheat with a bright blue LED flashlight and some orange safety glasses in a dark room. I've also noticed if you load a ton of DNA into it you can actually see a visible orange band under ordinary lighting but obviously that's not ideal for most applications.

Thank you so much Dr. Christoph Metzendorf for your advice.

@ I also use TAE buffer to make and run the gel electrophoresis.

@ For the rest of the advice, I will try to figure it out one by one and will update you as soon as possible.

@ For single PCR products, do you mean a single band after running the double digested products in gel electrophoresis????

@ So do you mean that we can use psicheck-2 vector right after the double digestion without doing the gel purification.

Thank you so much Alexandra Johnson for your advice.

I have tried to lessen the chaotropic salt during the gel extraction as you recommended. However, I still get a low A260/A230 ratio (DNA concentration: 17 ng/uL), so I guess perhaps the low concentration of DNA mostly contributes to low A260/A230 in this case.

Hi, I would recommend view changes that may help you to solve your cloning problem (especially #4):

1. Cut for a shorter period of time let say 1h.

2. Add CIP into the digestion mix- CIP does works in most restriction buffers.

3. Check for the presence of restriction sites in the PCR product, if they are introduced during amplification check your primers, you must have few additional nucleotides depending on the restriction sites.

4. At last but maybe most important: After CIP treatment of the vector part you must do Phenol: chloroform extraction. CIP is a thermostable enzyme and its residual activity after gel extraction will kill your ligarion.