PGem Easy Vector System: ampicillin conc and plasmid length problem - (Sep/20/2010 )

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.

http://img718.imageshack.us/img718/1376/iy003959copy.png

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)?

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?

moerae on Tue Sep 21 02:34:49 2010 said:

HomeBrew on Tue Sep 21 03:00:40 2010 said:

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?

hianghao on Tue Sep 21 07:43:20 2010 said:

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

hianghao on Mon Sep 20 08:29:10 2010 said:

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.

HomeBrew on Tue Sep 21 09:12:27 2010 said:

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...

hianghao on Tue Sep 21 13:56:29 2010 said:

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

hianghao on Tue Sep 21 13:56:29 2010 said:

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.

perneseblue on Wed Sep 22 00:41:41 2010 said:

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...