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Subcloning into pMIR-REPORT-luc


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#1 abyrd98

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Posted 21 September 2009 - 10:58 AM

Hey everyone, I'm about to pull my hair out over this and am hoping to find some help here. I'm trying to use Ambion's pMIR-REPORT-luc reporter plasmid to clone in a 40bp insert in the 3' UTR of the luciferase gene. The restriction sites are SpeI and HindIII and are located 62bp from each other. I've conducted around 6 digestion, ligation and transformation experiments varying around the protocol below and I get ZERO colonies or the colonies that have grown (1 or 2 on a plate...at best) do not contain my plasmid. PLEASE HELP!

I'm using a dsDNA oligo "Insert" I order from IDT w/sticky ends and 5' phos. I set up a double digestion of my vector as follows:
dH2O= 32.55uL
10x Buffer B (Promega)= 4uL
BSA (10ug/uL) = .4uL
Vector (1.9ug/uL) = 1.05uL (2ug)
---mix by pipetting, then (+):
SpeI (10u/uL) = 1uL
HindIII (10u/ul) = 1uL
Tot. Rxn Vol = 40uL

I incubate this at Room Temp for 3hrs then Gel Purify the band after running on agarose gel. I use the GeneClean Glassmilk beads for this. Everything looks good up to this point. My gel has a single band at the right size for digested vector. I've included SpeI only and HindIII only controls for pMIR vector as well as pBMN and both enzymes are digesting.

For my ligation I'm using T4 DNA Ligase with T4 Buffer. I've been using 100ng of vector and have tried Insert:Vector ratios of 1:1, 2:1, 3:1, 4:1, 10:1, 15:1, 20:1, and 30:1. All of these have been in 20uL Rxn volume and conducted at RT for 3hrs (except 1 where I ran it at 4oC overnight).

I use standard tranformation protocol for XL-1 Blue E.coli: Thaw (+) DNA Rxn directly to E.coli--> leave on ice 20min--> heat shock 42oC 90sec--> ice for 1 min --> (+)300uL LB and shake for 45min @ 37oC then streak on Amp plates and grow up overnight @ 37oC.

I've included positive ctrl of pMIR-REPORT-luc with NO digestion and get tons of colonies. I've done neg control of double digested vector (-) insert and I get zero colonies. I've included a BlpI restriction site in my insert (there's not one in the rest of the vector) for a diagnostic digest. The few times I've gotten colonies (every time its been in 4:1 and/or 10:1 Rxn) the BlpI digestion of a mini-boiling prep does not give me the right band size. Typically I've been getting two bands: one around the right size and one about 3kb smaller.

I have NO idea what's going on. This should NOT be this complicated. ANY thoughts would be appreciated.

Thanks,

Andy

Edited by abyrd98, 21 September 2009 - 10:59 AM.


#2 Warren

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Posted 21 September 2009 - 01:14 PM

Hey everyone, I'm about to pull my hair out over this and am hoping to find some help here. I'm trying to use Ambion's pMIR-REPORT-luc reporter plasmid to clone in a 40bp insert in the 3' UTR of the luciferase gene. The restriction sites are SpeI and HindIII and are located 62bp from each other. I've conducted around 6 digestion, ligation and transformation experiments varying around the protocol below and I get ZERO colonies or the colonies that have grown (1 or 2 on a plate...at best) do not contain my plasmid. PLEASE HELP!

I'm using a dsDNA oligo "Insert" I order from IDT w/sticky ends and 5' phos. I set up a double digestion of my vector as follows:
dH2O= 32.55uL
10x Buffer B (Promega)= 4uL
BSA (10ug/uL) = .4uL
Vector (1.9ug/uL) = 1.05uL (2ug)
---mix by pipetting, then (+):
SpeI (10u/uL) = 1uL
HindIII (10u/ul) = 1uL
Tot. Rxn Vol = 40uL

I incubate this at Room Temp for 3hrs then Gel Purify the band after running on agarose gel. I use the GeneClean Glassmilk beads for this. Everything looks good up to this point. My gel has a single band at the right size for digested vector. I've included SpeI only and HindIII only controls for pMIR vector as well as pBMN and both enzymes are digesting.

For my ligation I'm using T4 DNA Ligase with T4 Buffer. I've been using 100ng of vector and have tried Insert:Vector ratios of 1:1, 2:1, 3:1, 4:1, 10:1, 15:1, 20:1, and 30:1. All of these have been in 20uL Rxn volume and conducted at RT for 3hrs (except 1 where I ran it at 4oC overnight).

I use standard tranformation protocol for XL-1 Blue E.coli: Thaw (+) DNA Rxn directly to E.coli--> leave on ice 20min--> heat shock 42oC 90sec--> ice for 1 min --> (+)300uL LB and shake for 45min @ 37oC then streak on Amp plates and grow up overnight @ 37oC.

I've included positive ctrl of pMIR-REPORT-luc with NO digestion and get tons of colonies. I've done neg control of double digested vector (-) insert and I get zero colonies. I've included a BlpI restriction site in my insert (there's not one in the rest of the vector) for a diagnostic digest. The few times I've gotten colonies (every time its been in 4:1 and/or 10:1 Rxn) the BlpI digestion of a mini-boiling prep does not give me the right band size. Typically I've been getting two bands: one around the right size and one about 3kb smaller.

I have NO idea what's going on. This should NOT be this complicated. ANY thoughts would be appreciated.

Thanks,

Andy


You should incubate your restiction digestion at 37 (that may be a misprint) but if I understand you correctly, you basically have two oligos, that when annealed will have 4 base overhangs that will ligate to your restriction sites, right? I have done something similar recently. One thing you want is to get your oligos annealed nicely, which can be accomplished by heating to boiling or thereabouts (over 90) and then slow cooling. Your double digestion seems to work given your control with no insert. The other trick here I would suggest is NOT phosphorylating the oligos -- if you do not dephosphorylate your vector, your insert doesn't need the 5' phosphate. You are making a very "easy" ligation difficult, by having phosphorylated tiny fragments, which will preferentially concatamerize, and making it difficult to get only one to ligate to both of your vector sites. Even if you get it to work, you'll probably end up with a bunch of clones with multiples of the insert. Dephosphorylate your oligos, anneal them as I suggest, and I bet it will work like gang-busters on the first try. By doing it this way there is only one correct way the vector and insert can be ligated (just don't dephos your vector)! Good luck! Warren..

#3 abyrd98

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Posted 21 September 2009 - 04:02 PM

[/quote]

You should incubate your restiction digestion at 37 (that may be a misprint) but if I understand you correctly, you basically have two oligos, that when annealed will have 4 base overhangs that will ligate to your restriction sites, right? I have done something similar recently. One thing you want is to get your oligos annealed nicely, which can be accomplished by heating to boiling or thereabouts (over 90) and then slow cooling. Your double digestion seems to work given your control with no insert. The other trick here I would suggest is NOT phosphorylating the oligos -- if you do not dephosphorylate your vector, your insert doesn't need the 5' phosphate. You are making a very "easy" ligation difficult, by having phosphorylated tiny fragments, which will preferentially concatamerize, and making it difficult to get only one to ligate to both of your vector sites. Even if you get it to work, you'll probably end up with a bunch of clones with multiples of the insert. Dephosphorylate your oligos, anneal them as I suggest, and I bet it will work like gang-busters on the first try. By doing it this way there is only one correct way the vector and insert can be ligated (just don't dephos your vector)! Good luck! Warren..
[/quote]

Thanks for the feedback. This will work out good as I need to do a double digestion of my vector tomorrow. I'll be sure to not dephos. Also, the insert is suppose to already be annealed with the overhangs. You think I should dephos the dsDNA insert, then heat and reanneal just to be safe?

#4 phage434

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Posted 21 September 2009 - 04:20 PM

You'll only favor multimers with high molar concentration of the oligos relative to your vector. If you do that calculation correctly, multimers should not be a problem.

#5 Warren

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Posted 21 September 2009 - 05:51 PM

[/quote]

You should incubate your restiction digestion at 37 (that may be a misprint) but if I understand you correctly, you basically have two oligos, that when annealed will have 4 base overhangs that will ligate to your restriction sites, right? I have done something similar recently. One thing you want is to get your oligos annealed nicely, which can be accomplished by heating to boiling or thereabouts (over 90) and then slow cooling. Your double digestion seems to work given your control with no insert. The other trick here I would suggest is NOT phosphorylating the oligos -- if you do not dephosphorylate your vector, your insert doesn't need the 5' phosphate. You are making a very "easy" ligation difficult, by having phosphorylated tiny fragments, which will preferentially concatamerize, and making it difficult to get only one to ligate to both of your vector sites. Even if you get it to work, you'll probably end up with a bunch of clones with multiples of the insert. Dephosphorylate your oligos, anneal them as I suggest, and I bet it will work like gang-busters on the first try. By doing it this way there is only one correct way the vector and insert can be ligated (just don't dephos your vector)! Good luck! Warren..


Thanks for the feedback. This will work out good as I need to do a double digestion of my vector tomorrow. I'll be sure to not dephos. Also, the insert is suppose to already be annealed with the overhangs. You think I should dephos the dsDNA insert, then heat and reanneal just to be safe?
[/quote]

The annealing is so easy to perform, I would just go ahead and dephos and reanneal, but its up to you. All you have to do is heat up a heat block, then shut it off, or boil a beaker of water an let it cool to room temp. I recently did this with something even a bit harder where I was making about 100 bp inserts so used 6 oligos per insert and only phosphorylated in inner oligos (to make the insert) and made sure to leave the outside oligos where the overhang will be dephosphorylated. I made 16 different constructs that way and cloned them into multiple vectors, and it worked perfect the first time every time, for every 16 or so minipreps I might get one that was not correct.

Warren..

#6 Warren

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Posted 21 September 2009 - 05:59 PM

You'll only favor multimers with high molar concentration of the oligos relative to your vector. If you do that calculation correctly, multimers should not be a problem.


There may be a "perfect" ratio you could hit, but with such a tiny insert, its going to be hard to find conditions that will allow that "perfect" construct, when everything is phosphorylated and can ligate together in many ways. The way I am suggesting, the insert/vector ratio ends up not being too critical at all , because the only things that can ligate are the correct construct and the vector to itself in multimers. All other overhangs will not be ligated and "fall apart" until they anneal correctly (they way you want)! Its alot easier (to me) than playing with ratios! Warren..

#7 Warren

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Posted 21 September 2009 - 07:29 PM

[/quote]

You should incubate your restiction digestion at 37 (that may be a misprint) but if I understand you correctly, you basically have two oligos, that when annealed will have 4 base overhangs that will ligate to your restriction sites, right? I have done something similar recently. One thing you want is to get your oligos annealed nicely, which can be accomplished by heating to boiling or thereabouts (over 90) and then slow cooling. Your double digestion seems to work given your control with no insert. The other trick here I would suggest is NOT phosphorylating the oligos -- if you do not dephosphorylate your vector, your insert doesn't need the 5' phosphate. You are making a very "easy" ligation difficult, by having phosphorylated tiny fragments, which will preferentially concatamerize, and making it difficult to get only one to ligate to both of your vector sites. Even if you get it to work, you'll probably end up with a bunch of clones with multiples of the insert. Dephosphorylate your oligos, anneal them as I suggest, and I bet it will work like gang-busters on the first try. By doing it this way there is only one correct way the vector and insert can be ligated (just don't dephos your vector)! Good luck! Warren..
[/quote]

Thanks for the feedback. This will work out good as I need to do a double digestion of my vector tomorrow. I'll be sure to not dephos. Also, the insert is suppose to already be annealed with the overhangs. You think I should dephos the dsDNA insert, then heat and reanneal just to be safe?
[/quote]

Now that I give it some more thought, if you are going to have to heat inactivate your phosphatase, I would actually go ahead and do the annealing step.....if you heat it up to dephosphorylate, you might as well go all the way and denature and anneal, it shouldn't hurt anything unless your sequences can anneal in multiple ways (not likely unless there is repeats). This will kill two birds with one stone, in case your oligos are not annealed correctly as they stand. Just cool slowly, and they'll anneal nicely (you can even see it on a gel, although 50 bp is tiny, you usually have so much oligo from a single batch that you can anneal enough to easily see on a gel). Good luck, Warren..

#8 losybelle

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Posted 30 January 2012 - 11:40 PM

Hello:

i've been trying to use the very same vector and my problem is that even if I'm getting cellswith the clone, all of the have any problem. The first time i got them i lost the luciferase gene, the second time i lost the CMV and two days ago i find out two plasmid which had like another extra 3kb in their sequence.

Does anyone have an idea about what's going on? i thought that maybe it would be a problem with the compatibility of the e.coli strains i was using, maybe something about recombination. I've used JM109 from promega the first time, and now i was using DH10B. I haven't found any restrictions about the e.coli strains in the manual.

Thanks




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