Dear Praveesh

Which cloning approach are you using?

TOPO cloning or standard restriction enzime cloning? And are you using enzimer generating BLUNT or Sticky ends?

Ciao

Manuele

Hi there,

If insertion was random and the orientation had no effect on cell viability after transformation, you would indeed expect a 50% probability of getting the right orientation on each clone. If the supposition is considered right it means you would have roughly 1/1000th to get 100% wrong orientation in a sample of 10 (0.510) which is very bad luck. So there must be something else... My feeling is that your target protein, if expressed in cell, might compromise cell viability. Therefore the right orientation while cloning might be a lethal event after transformation due to the toxicity properly expressed gene. So my questions are: What is your target? What plasmid do you use? Has it a leaky expression in a cloning strain?

Remark: 10 is not a lot. It could be worth trying to get more...

As a control experiment, you could try a cloning with 2 different sites...

It may work better in the right direction, but it could in any. It is not a mistake if it is in the opposite way, but it is not optimal. Make sure elements are in the right way, in the same way

In addition to other answers, be sure you're not recovering clones for too long prior to selection. They will multiply and lead to screening of daughter cells.

If you're cloning with sticky ends, there is often preference for direction; but its not likely with blunt end cloning.

Manuele Martinelli Its standard cloning with restriction enzymes by sticky ends

Dominique Liger. What you said makes sense but the vector expresses only in mammalian systems. The expression promoter is derived from a Plasmodium operon so it shouldnt express anything in the bacteria. Nonetheless, I agree that 10 clones is not much. I think I'll change all the enzyme batches and start from scratch with PCR amplification of the insert. Maybe somewhere along the line I did something initially that created this bias. Will use another plasmid with the same restriction site as a control.

Andrew Varley Yeah, It seems like there might be a preference for direction. But I wonder why its there.

Hi Praveesh Valissery

two questions:

1. which restriction enzyme do you use?

2. how do you prepare PCR insert? Which polymerase do you use in a PCR? Do you use restriction enzyme (RE) on the PCR product before ligation?

Mirjana Polovic

1. I used Xho1 from NEB

2. The insert was amplified using Phusion polymerase. After cleanup, I digested about 2 ug of the insert using Xho1 for 3h at 37C. The digestion was run on a gel and the band was cut out and cleaned using the kit and eluted in water. I quatitated the elution and performed the ligation (1:7, vector:insert molar ratio). The insert is about 150bp and the vector is 6.5 kb.

Praveesh Valissery

Okay. I need another information. How do you check for the orientation of the insert?

Mirjana Polovic Two ways :

1. The insert has a single asymmetric Pst1 site. Another site is on the vector. Digestion with this enzyme gives a unique sized fallout depending on the orientation of the insert.

2. I have a set of primers for diagnostic PCR : forward primer on the vector backbone and a reverse primer on the insert in the right orientation.

Dear Praveesh

if you use a single digestion enzime, also if it is a sticky end, is quite normal that you have aorund 50% or probability that it insert in the correct direction, because there is nothing that forse the direction of your ligases.

If you would like to solve this problem you need to perform double digestion, with sticky end enzimes and add 2 different enzime digestion site at the 3' and 5' of your insert.

Ciao

Manuele

Praveesh Valissery ,

everything seems alright in your steps.

Can you take a restriction enzyme that makes blunt-ends - ONLY for cutting your VECTOR?

Because your polymerase used in a PCR will produce blunt-end PCR product and then it should ligate without problems into a blunt-end vector. Then you do diagnostic digest as you mentioned with PstI.

Hopefully that works. Good luck!

Manuele Martinelli Unfortunately I do not have a MCS with mutiple enzymes to clone into. I agree that nothing should exist to force the insert in one direction. I just had an unsual result and was hoping someone else might have noticed something similar. I guess its just bad luck and will keep trying till it works. Thanks anyway.

Mirjana Polovic I'll try this blunt end cloning if the next round doesnt work. Thanks for your input.

Dear Praaveesh

you can use a different approach to insert the enzime that you would like.

Perfrom a vector PCR, with primer contaning the enzimes that you would like to use, digest the vector PCR and ligate with digested fragment.

Otherwise you can use an enzime cloning free approach (i like a lot the PIPE cloning)

if you are interested more details about if, you can check the following videos

https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU

https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8

that are available on my BLOG PRoteoCool

https://proteocool.blogspot.com/p/index.html

Ciao

Manuele

You could try using a TOPO-TA cloning kit. You can directly clone in ANY purified DNA with an A overhang. It's a bit expensive, but it works really well.

Hi Praveesh,

In theory there is 50% likelihood for each orientation but.... having some experience with cloning, i can confirm that there is always a preferred “orientation”... the solution is to screen more colonies. make sure you enrich your transformation with constructs carrying the insert, by dephosphorylating the vector after digestion and before ligation.. then you can do “colony PCR” with a primer in the vector and one in the insert, so that only the correct orientation will allow amplification (you can also try primer pairs for both orientations, so that you can check that either one pair or the other gives you the expected result). If you need further help, let me know.. and good luck!!

Manuele Martinelli Thanks for the suggestion. your blog was informative.

Katie A S Burnette I havent used TOPO kits yet, but from what I've read, I'll have to restrict the cloning to vectors that come with the kit. Since the vectors in the kit come covalently linked to a Toposimerase, the use of custom designed vectors in the lab would be impossible.

Paola Campomenosi I agree, I guess its a matter of luck. Increasing the vector amounts and screning more colonies would be the the best option. Thanks.

Try NEB assembly kit, easy to clone in correct orientation

An older method for those of us from the days when sequencing was much more expensive was to design a primer for your vector in the ideal orientation as the 5prime oligo and then a 3prime oligo antisense in your insert. Then just screen by colony PCR using a cheap Taq. You can integrate this strategy into any future projects so that you already have the primer pair ready to go. Good luck!