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PGem Easy Vector System: ampicillin conc and plasmid length problem


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10 replies to this topic

#1 hianghao

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Posted 20 September 2010 - 12:29 AM

I've cloned my inserts (270bp) into your PGem Easy Vector Sys.
At 1st, i used 10ug/ml ampicillin (according to neighbour lab protocol) and there were growth of more than 100 white colonies and only 2-4 blue colonies. I've selected 20 clones and did a miniprep, followed by gel electrophoresis. And, i found no band at all!! Control plates were good. I figured that it might due to the low conc of ampicillin.
Then, i used 100ug/ml ampicillin as recommended by PGem protocol, and plated on 10 plates. 6 plates showed no growth at all, another 4 plates with 2-4 colonies each, with only a total of 4 white colony. What was the problem (the dose was recommended by its manufacturer)? I proceeded the white colonies with minipreps, followed by gel electrophoresis.

Posted Image

As you can see, the plasmid bands were ~2.5kb. And from the product's detail, the plasmid is suppose to be 3kb long. Even if the plasmid contained no insert, it shouldn't be 2.5kb! Any idea.

P/s: i saw a small blue dot in the middle of some white colonies. Were those colony suppose to be blue or white? How come there was a blue dot in the colony? Was it because i incubated the plate too long (16-20h)?

#2 moerae

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Posted 20 September 2010 - 06:34 PM

Hi,

I use the pGEM-T easy system and haven't run into any major issues so far. I use even higher concentration of amp (150 ug/mL) and I tend to get an all right number of colonies. When I first used the system it worked really well. Alot of white colonies and very few blue ones. As for your gel, what kind of ladder are you using? Is it a supercoiled ladder or just normal DNA ladder?

#3 HomeBrew

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Posted 20 September 2010 - 07:00 PM

It looks as though you're comparing supercoiled (uncut) plasmid to a linear standard -- you can't estimate size accurately that way. If the bulk of your plasmid prep is supercoiled, it will migrate through the gel more quickly than a same-sized linear band. Cut your plasmid with a restriction enzyme(s) that will either linearize the plasmid (cut it once) or that will cut out your insert from the vector, then run the gel against the linear ladder. What samples are in the first two lanes?

#4 hianghao

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Posted 20 September 2010 - 11:43 PM

Hi,

I use the pGEM-T easy system and haven't run into any major issues so far. I use even higher concentration of amp (150 ug/mL) and I tend to get an all right number of colonies. When I first used the system it worked really well. Alot of white colonies and very few blue ones. As for your gel, what kind of ladder are you using? Is it a supercoiled ladder or just normal DNA ladder?



It looks as though you're comparing supercoiled (uncut) plasmid to a linear standard -- you can't estimate size accurately that way. If the bulk of your plasmid prep is supercoiled, it will migrate through the gel more quickly than a same-sized linear band. Cut your plasmid with a restriction enzyme(s) that will either linearize the plasmid (cut it once) or that will cut out your insert from the vector, then run the gel against the linear ladder. What samples are in the first two lanes?


I see... I didn't realise that i need a supercoiled ladder! I don't think i have it in my lab! Btwy the first two lanes were positive control for 5' and 3' RACE.

I did another cloning on 50ug/ml plate and incubated for 20 hours. There were more than 50% blue colonies. Was it because i incubated it for too long?

#5 HomeBrew

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Posted 21 September 2010 - 01:12 AM

I see... I didn't realise that i need a supercoiled ladder! I don't think i have it in my lab!


But you do have restriction enzymes, right? So you can linearize your plasmid rather than using a supercoiled ladder?

#6 perneseblue

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Posted 21 September 2010 - 02:32 AM

P/s: i saw a small blue dot in the middle of some white colonies. Were those colony suppose to be blue or white? How come there was a blue dot in the colony? Was it because i incubated the plate too long (16-20h)?


Those are colonies which poorly expresses lacZ. Sometimes a plasmid will capture a DNA fragment, usually a small fragment, in frame with the Beta unit of the lacZ gene, giving rise to a poorly functioning fusion protein.

On the rare occasions this type of colonies are your positives (you can check the DNA sequence on your plasmid manager program to see if the lacZ gene is inframe), but nearly all the time it isn't.
May your PCR products be long, your protocols short and your boss on holiday

#7 hianghao

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Posted 21 September 2010 - 05:56 AM


I see... I didn't realise that i need a supercoiled ladder! I don't think i have it in my lab!


But you do have restriction enzymes, right? So you can linearize your plasmid rather than using a supercoiled ladder?


I don't have RE either...
Can i do a pcr using my primer to check whether the insert was inside? I think i should have run a colony pcr before miniprep...

#8 HomeBrew

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Posted 21 September 2010 - 11:14 AM

I don't have RE either...
Can i do a pcr using my primer to check whether the insert was inside? I think i should have run a colony pcr before miniprep...


Yes. You can do a PCR on your purified plasmid DNA (that you already have) as well.

#9 perneseblue

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Posted 21 September 2010 - 04:41 PM



I see... I didn't realise that i need a supercoiled ladder! I don't think i have it in my lab!


But you do have restriction enzymes, right? So you can linearize your plasmid rather than using a supercoiled ladder?


I don't have RE either...
Can i do a pcr using my primer to check whether the insert was inside? I think i should have run a colony pcr before miniprep...

If you do run a colony PCR, remember to amplify across the junction between the insert and vector (ie one primer binds to the vector and the other the insert) PCR is very sensitive and will readily amplify naked DNA sitting the agar plate, leftovers from the ligation reaction.
May your PCR products be long, your protocols short and your boss on holiday

#10 hianghao

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Posted 23 September 2010 - 02:55 AM




I see... I didn't realise that i need a supercoiled ladder! I don't think i have it in my lab!


But you do have restriction enzymes, right? So you can linearize your plasmid rather than using a supercoiled ladder?


I don't have RE either...
Can i do a pcr using my primer to check whether the insert was inside? I think i should have run a colony pcr before miniprep...

If you do run a colony PCR, remember to amplify across the junction between the insert and vector (ie one primer binds to the vector and the other the insert) PCR is very sensitive and will readily amplify naked DNA sitting the agar plate, leftovers from the ligation reaction.


I see... I've ran colony pcr using my original primers that created the insert. Then i've selected those with band for minipreps. No wonder some of my minipreps showed bands that are shorter than others. The colony pcr must have amplifiedt he naked DNA instead...

#11 HomeBrew

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Posted 23 September 2010 - 03:34 AM

If you have a primer that binds to the vector and one that binds to the insert, always use that set if you're going to do PCR on colonies directly from the transformation plate. Alternatively, you can pick the transformants from the transformation plate to a fresh plate, grow that overnight, and do colony PCR using growth from the fresh plate as template. If you pick your transformants first, you can use either two primers that target the insert or one that binds to the vector and one that binds to the insert.

In either case, note that using a primer that binds to the vector and one that binds to the insert is orientation specific -- if you use a vector borne forward primer and an insert borne reverse primer, clones that contain the insert in the opposite orientation will not amplify. If the orientation of your insert is important, this can be a good thing -- you get both pieces of data (the presence of the insert and its orientation) simultaneously. If orientation of the insert is irrelevant, you'll miss some positive clones unless you do two PCRs on each. Using two insert-specific primers avoids this problem, but you must patch you colonies to fresh media first to avoid false positives.




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