Sunday, April 18, 2010

Troubleshooting Gateway Cloning

After struggling with a particularly annoying problem in the lab, I finally got my experiment to work this weekend! When I crowed about this to my partner, he told me that whenever he figures out the solution to a tricky problem in his line of work, he blogs about it, for the sake of others who might have the same issue. I did my share of Googling while troubleshooting my experiments and didn't find a solution, so I thought this was worth blogging about. The following will make very little sense if you don't do molecular cloning.

I spent almost three months trying to clone my DNA sequence of interest into a mammalian expression vector using Invitrogen's Gateway Cloning system. Things were going smoothly for a while -- I made a pENTR entry clone, chose a pDEST destination vector, did the LR clonase reaction according to the instructions in the kit, did a diagnostic restriction digest to confirm that my plasmid had the expected sequence, and then... I couldn't maxi-prep the plasmid. This was my first time using the Gateway system, so I expected to hit some bumps in the protocol, but I've been doing maxi preps (with Qiagen kits -- regular and the HiSpeed) for years. So, I felt pretty stupid when my experiment broke down at that point. I got my plasmid out of a mini prep, but failed to recover the plasmid after inoculating a maxi-sized culture with the same exact construct in the same exact competent cells. What the hell?

As I grew increasingly frustrated, my adviser had the outlandish idea to repeat the experiment with good controls. So I did. I used every control I could think of. I transformed, mini-prepped, and maxi-prepped: my entry vector, a known entry vector with a similar sequence that we had lying around the lab, my expression vector, a similar expression vector from the lab, and the empty entry vector. I also repeated the LR clonase reaction with my plasmids and with other Gateway plasmids we had on hand.

I could get maxi prep DNA from everything except LR reaction products (i.e., any sequence of interest flipped from pENTR into pDEST through attR/attL recombination). For some reason, the expression clones would grow in mini preps, and some sort of antibiotic-resistant bacteria would still grow in my maxi cultures, but I would get abysmal plasmid DNA yield from the maxi preps. I tried growing my bacteria at a lower temperature over a longer amount of time, which sometimes helps bacteria keep larger plasmids (my plasmid of interest was about 9 Kb), but that didn't help. I bought fresh new LR clonase enzyme, but that didn't help either.

Eventually, senior lab members remembered someone having a similar problem a few years ago. They thought that the issue was finally resolved by making fresh pDEST vector by culturing bacteria containing the empty vector from the original glycerol stock. I thought that made no sense, but it was easy enough to dig the glycerol stocks out of the freezer, grow up some cells, and prep the DNA. So I did. I then used the newly made pDEST DNA to repeat the LR clonase reaction with my entry clones, transformed the LR product, and grew up some bacteria for a maxi prep.

And... it worked!

I don't know why it worked. I guess somehow, after being stored in the freezer for a while and going through enough freeze-thaw cycles, the pDEST DNA that we had stockpiled went bad. It worked well enough to take up the insert from my pENTR clones and grow in competent cells in a 2 mL culture, but then it petered out when I tried to grow a 150 mL culture of the same cells. So, if you are having a similar problem, I say to you: find your old glycerol stocks. My labmates say that the last time they had this problem, transforming the plasmid back into some ccdB-tolerant cells (the pDEST vector we used contains the ccdB suicide gene) and re-prepping from them also worked, but I can't verify that.

Next up: transfecting my expression vector into some mammalian cells to see what happens! (Also known as: the experiment that I actually care about, which I was sure I would have gotten to by now...!)

43 comments:

  1. So how about the pENTR? The thaw-freeze cycles on pENTR wouldn't affect the LR reaction???

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  2. Anonymous,

    As it turns out, my pDEST problems are far from over. While going back to the old stock of pDEST worked for a while, I'm still having problems. Now we think there might be an issue with our competent cells -- using commercial cells instead of our homemade ones seemed to help a lot. For some reason, the bacteria are losing the plasmid when I re-inoculate them to grow maxi prep cultures. Making a fresh batch of competent cells next week to see if that helps... Thanks for reminding me to update this post!

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  3. Hi,
    Look like you are using Lentiviral vectors (9kb) if so I had same problem. We wasn't able to get any clones from glycerol stock without plating them on dishes. It is not depend from LR, It is depend from large repeats. We had same problems even we used Restriction/Ligation protocol.

    We now making following:
    1. after LR reaction grow miniprep
    2. extract DNA by fast dna extraction protocol (Zymo Miniprep) from part of cells.
    3. Digestion
    4. seed for midiprep LB in same day with fist extraction.

    But it is not all! some more hints:
    1. Cultivate at 28C! Exist even paper where were compared different temperature and at 37 happens recombinations. In my experience, 30C is to high.
    2. Use LB media! Do not use SOC or others. Risk of recombination significantly higher.
    3. if possible use only Stbl3 compitent cells. In couple week we are going to try HB101 which is recommended for unstable vectors but we didn't try it yet.
    4. For cultivation cells on dishes use home made LB agar, do not use ImMedia and other fast in preparation mixtures. Last our comparasion for 3 clones were (home made vs ImMedia, good/total): 4 (good) from 4 (total) vs 2 from 4; 4 from 4 vs. 1 from 4; 3 from 4 vs. 0 from 4.
    5. If you have clone more faster will be make re-transformation than spend time with frozen stock.

    I have question to you:
    Have you ever try "reverse" BP?
    It is mean:
    1. LR with Dest-R4R2 + ENTR-L4R1 + ENTR-L1R5+ ENTR-L5L2 on Ampiciline dishes
    2. BP with DONR-P1P5r, ccdB survival cells, Chloramphenicol.

    I have problem with second BP. Do you have any idea how to make it?

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  4. I am using lenti and Gateway cloning. I have exactly the same problem! I am so glad to find your blog! This is the only place in the entire internet talking about this particular problem.

    Did you try to streak new plates every time you do maxiprep. It seems unnecessary, but it might be important for unstable plasmids. Two of my labmates have done maxiprep from the same glycerol stock. One streaked a plate and picked a colony for maxiprep and the other one didn't. The result is the former succeeded and the latter didn't get anything.

    So I am not repeating the LR reaction. I am trying to streak a new plate for maxiprep right now.

    If you figured out anything new , please let me know. I am doing my first year postdoc training in UAB. My email is luowenyi@uab.edu.

    Thanks!

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  5. All of you seem to hace work a lot with gateway... so I hope you can help me with this:

    I don´t know if it is posible to make an LR reaction having an cDNA in a vector whose 260/280 relation is under 1.81 measuring in a nanodrop

    thanks

    Veronica

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  6. wow
    This is a wonderful blog for gateway cloning.
    Guys, I am working with this technology and found it easy. But some times i was not able troubleshoot the problems. I wasted 4 months with old BP and LR clonase enzymes. People are saying we can not thaw and use not more than 3 times. I ordered new clonase II and did my BP reaction. I got same restriction pattern but i will go for sequencing before doing LR.

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  7. Hi all

    Fariha here. Please help me and suggest something. I am facing a problem with BP reaction. I transformed BP reaction in DH5@ competent cells then spread on kanamycin selective plates (as my vector has resistance gene for this antibiotic )but I didn't get colonies overnight incubation but then almost after 30 hours colonies appeared. I did colony PCR and did't get my product band. Now I am planning to do plasmid miniprep again same problem growth of colony in liquid culture supplemented with kanamycin is very slow and might take again 30 hours. What's wrong here?

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  8. If your bacteria take 30 hours to grow, you are probably getting non-selective growth of cells that don't contain your plasmid. What I was told is that after many hours, the antibiotic can get "used up" from killing all the non-resistant bacteria and the selection goes away, so you are just growing any old bacteria. It sounds like you need to repeat your BP reaction and transform again. And maybe try using fresh BP enzyme, since it can become less effective if not stored properly.

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  9. Dear Laura

    Thanks a lot for the suggestion. Yeah your right about the antibiotic. Seems that my antibiotic got unstable. I think nothing is wrong with enzyme, actually enzyme clonase is stored at - 20 freezer. Its almost my first BP reaction that I have discussed here but surprisingly I got proper growth after overnight in positive and negative BP controls (showing that the enzyme is active). Still no idea why is this happening :(. I hope I get the plasmid tomorrow then I will discuss with you dear.

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  10. Hi to everyone and dear Laura

    Last time I discussed about failed BP reaction here. Please guide me about amount of DNA to be cloned in entry vector. Also guide me about the protocol to have an efficient BP and LR reaction. I will be very grateful.

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  11. Dear Laura

    I have tried sequencing pENTR minus the stop codon with M13 primers F and R, but i still wont get the sequence read(NNN). I have used other plasmids as controls and they seem to be okay. Any idea on how i should go about it?

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    Replies
    1. Hi,
      I had similar problem!
      I used pENTR/SD/D-TOPO, I can't get sequence read longer than 200bp(most of them is 50bp). then later when I check they all don't contain any insert. only positive was having 1kb insert. Yes, I was too optimistic in first try of TOPO cloning based on manual.

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  12. Hey, thanks for blogging on gateway cloning. I tried for my gene from entry construct to destination. I got colonies and picked a few for minipreps. Both candidates have shrunken plasmids. How often does this happen with gateway? A second problem is I tried to make more destination vector plasmids by transformation and mini-preps. Transformation worked well but the yield for plasmid from mini-prep is below 10ng/uL. This was of course with commercial ccdB survival competent cells. The sizes of the colonies look normal, just no plasmids. Any trick?

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  13. I used destination vector(PDS-CMV~) to transform ccdb survival competent cell( 'One Shot® ccdB Survival 2-T1R' from invitrogen).
    The yield for plasmid from miniprep is above 100ng/uL.
    But When I was doing BP reaction using my PCR sequence and PDONR221(invitrogen), The yield for plasmid from miniprep was below 100ng/uL.

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  14. trying changing fresh vector and bp clonase.. repeated freeze thaws makes it go bad.. best thing to do is keep them in aliquots..

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  15. i used destination vector of topo clonig all was fine with the cloning part and i got the correct sequence of my clone but the part where i feel difficult was about the expression of protein in the expression strain of BL21-AI provided with the kit . i tried to optimise for temperature and arabinose conc. for the protein expression but could not get the expression of my protein nor even the +ve control of gus in penter vector.

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  16. is there arabinose to do with the expression of protein with the vector if it is to be expressed in some other cell like BL21 -RIL ,or bl21 -si

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  17. what would be the suitable host for expression of protein cloned in destination vectors as pdest 14,pdest 15,17 or42. as i have tried all the vetors in the host cell bl21-ai but not got the protein expression

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  18. Hi to all,

    My Bp reaction is not working , although i have repeated it two times and this is the first time im using gateway tech. please can someone suggests me what should i do. it worked fine throughout the process but when i checked for the right size . i cannot see any band . although the over night cultur shown alot of bacterial growth. and the plating seemed fine too shows reasonable growth

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    Replies
    1. miss karim my BP reaction is also not working...just wondering did yours finally work?if so then what did you do?

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    2. miss karim my BP reaction is also not working...just wondering did yours finally work?if so then what did you do?

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  19. hey guys,,this is a wonderful bolg for gateway troubleshooting. I am having a real big issue with gateway cloning, would love to take your help/suggestions for the same:
    I followed invitrogen's protocol and everything worked fine till i did my LR reaction. The colonies on my plate post lr reaction are heterogenous i.e there are small and big colonies. When I did a digest using EcoNI I can see multiple bands on my gel and more importantly I can see bands corresponding to my BP product in the LR clone . Does anyone know a way to solve this? I have already done a positive control to test the clonase enzymes. Pleaze help !
    R

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  20. I Have problem with LR. I am using phellsgate12 vector as expression vector, PB reaction worked well and I got pENTR, but can not get any colony in LR. in some case I got some colonies but PCR screening and digestion failed! please advise me!

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    Replies
    1. I got the same problem. Actually, I did lots of gateway cloning: 75, 2 of them does not grow at all after LR recombination. p223 Entry clone of these two grows well, and the rest LR reaction works well too.

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  21. Hi I Did LR reaction using pDEST-17 vector and also with PEG203 vector ,I get many colonies after transformation,but when I do colony pcr I don't get my band....Please help me!!

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  22. Has anyone ever had an issue propagating pDEST-R3-R4? When we transform DNA into One Shot® ccdB Survival™ 2 T1R Competent Cells and grow the transformation on Amp/Camp plates and then miniprep colonies from the transformation grown in media containing Amp/Camp (all at 37 degrees C or at 30 degrees C), most do not show the expected restriction digest pattern. Additionally, when we sequence, the ccdB gene is often mutated. Any help or suggestions are appreciated!

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  23. I'm also having problems with the LR reaction. Has anyone tried linearizing their pENTR vector yet?

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    Replies
    1. I've tried LR reaction with PCR product from entry vector using M13 primers. I got 100% transformation rate based on colony PCR with vector-specific forward and gene-specific reverse primers. However, after plasmid prep, I repeated PCR using the same primers and restriction digested the plasmids, but I don't see my insert. Something went wrong... any idea?

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  24. I m preparing a cdna library from a cell line I m using gateway cloning using pdonr and evrogen c dna library kit. when I amplify c dna library with att b adapters I get a smear. I dont know is that what comes when I do att b pcr with my ds cdna prepared by evrogen kit. Problem is there is very strong secondary structure formation. How to prevent that.. so that optimum amplification

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  25. I have done LR recombination using pENTR/D-TOPO and pHellsgate12, when i perform PCR i can see fragment of my insert but restriction digest and sequecing with 35S primer did not yield results. What might be the problem and how should i solve the problem.

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  26. Hi. What I am about to aak may sound silly, but I am new to molecular biology. I have an empty vector pcdna3.1 that I wanto to transfect into mammalian cells as my control. The question is, how do Istart? Do i transform the pcdna into bacteria competent cells, streak and innoculate, then midi- or maxiprep it? Or can I just transfect my cells with the pcdna i have now?

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  27. Hmm, good to find out that I am not the only person dealing with this issue.

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  28. I met a problem with BP reaction. In fact, I have being tortured by the problem for several months. When I did the BP reaction between my genes (about 20 genes) and pDONR221, and transformed the BP products into DH10B, among those 20 BP products, one always does fail to grow, that is, growing very slowly and abnormally or resulting in very big clones. So, I think, the pDONR221 is good and the whole system that I used is fine. Minipreping from the abnormal clones, the size of the plasmid is not right, and sequencing it, no right sequence could be seen, only with broken fragments. I repeated it again and again, all do not work. Recently, I repeated it again, haha, no clones could be seen anymore.

    Do you have any comments on it?

    Any suggestions would be appreciated.

    Please helping me clearing this problem.

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  29. Hi, I have a problem with my pENTR/D-TOPO cloning. Almost all of my colonies are showing backward orientation of my gene of interest. I checked my reverse oligo and it doesnt seem to have any obvious binding with the 5' CACC of my forward oligo. Has anyone seen this problem before? If so, any suggestions please.....

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  30. Hi
    I am facing problem after BP reaction of pDONR221 and attB PCR product.
    I got lot of colonies after transformation of BP reaction product and they seem to gene and insert length specific suggesting reaction working.
    I am growing these colonies in the 1 ml overnight culture with the kanamycin (new).
    Problem is coming when I am doing the mini prep from these cultures. I am not getting any plasmid after mini prep. Tried both Qiagen and Clontack mini prep kits.

    I am doing all experiments with the recommendations of Invitrogen. New kits. New Plasmid.

    Thanks for your suggestions


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  31. Hi
    I am facing problem after BP reaction of pDONR221 and attB PCR product.
    I got lot of colonies after transformation of BP reaction product and they seem to gene and insert length specific suggesting reaction working.
    I am growing these colonies in the 1 ml overnight culture with the kanamycin (new).
    Problem is coming when I am doing the mini prep from these cultures. I am not getting any plasmid after mini prep. Tried both Qiagen and Clontack mini prep kits.

    I am doing all experiments with the recommendations of Invitrogen. New kits. New Plasmid.

    Thanks for your suggestions

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    Replies
    1. Hi SP, Did you check your colonies by direct colony PCR to see if they have your gene of interest? If so do you see any smear on the gel after PCR suggesting degradation?

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    2. I tried that also but same fail result.

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    3. Ok SP. if you don't see your band of interest after colony PCR then that could mean one of two things
      1) For some reason PCR is not working. Sometimes it happens for colony PCRs. (make sure you are mixing the colony cells with the PCR mix very well with pipette)
      2) The colonies that you are getting don't have your gene of interest.
      But if second case is true then that doesn't explain getting lot of colonies after transformation. Can you tell the approximate number of colonies you got after transformation, the volume you plated and the concentration of Kanamycin you are using?

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  32. Thanks
    I must have tried many mini preps directly from colonies. None of them have plasmid. Not even empty.
    Then I set up the positive and negative control provided with the kit. I got same result with unlimited colonies without plasmid. Negative was fine without any colony but positive have many-2 colonies. All are empty.

    Colonies.. No empty space in plate and plates If I spread it to more plates.

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    Replies
    1. If you are using PCR and restriction digest, then you definitely don't have your gene there. However, I would check your primers to make sure they are absolutely correct, keep in mind you need those flanking regions for insertion into pDONR.

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  33. Has anyone tried the gateway reaction at 37 degrees?

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    Replies
    1. The enzymes in clonase work best at RT - so it should still somewhat work, but your efficiency is going to tank.

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