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Cloning - False positive results - HELP!! (Jul/15/2008 )

Hi

I have a 450bp sequence that am trying to insert into a 2.9KB vector. Although I get colonies on the plate, when I try isolating the plasmid I cant see any plasmid. Can anybody help?

Here are the details...

I conduct a PCR and get my insert from a chrom. DNA - Et.OH precipitation - Restriction digestion - Then run it all in a gel - gel extract - set up 5 min Quick ligation (Sigma's QuickLink DNA ligation kit) - then a 2ul transformed into 50ul competent cells - heat shock - add 250ul of SOB - incubate 1 hr - spread plating on plates with ampicillin.

On incubation overnight I saw colonies - picked them - conducted plasmid isolation. - then ran a small volume in gel but cant see any plasmids.

Now am lost.

-Shiv8-

QUOTE (Shiv8 @ Jul 15 2008, 02:05 PM)
Hi

I have a 450bp sequence that am trying to insert into a 2.9KB vector. Although I get colonies on the plate, when I try isolating the plasmid I cant see any plasmid. Can anybody help

This kind of problem (basic) is best to troubleshoot with a senior person in the lab. There are several little things that may have gone wrong. While you wait till some forumite helps you out, talk with other people in and out of the lab. Serious advice, serioulsy. this will go a long way.

-cellcounter-

QUOTE (Shiv8 @ Jul 15 2008, 01:05 PM)
Hi

I have a 450bp sequence that am trying to insert into a 2.9KB vector. Although I get colonies on the plate, when I try isolating the plasmid I cant see any plasmid. Can anybody help?

Here are the details...

I conduct a PCR and get my insert from a chrom. DNA - Et.OH precipitation - Restriction digestion - Then run it all in a gel - gel extract - set up 5 min Quick ligation (Sigma's QuickLink DNA ligation kit) - then a 2ul transformed into 50ul competent cells - heat shock - add 250ul of SOB - incubate 1 hr - spread plating on plates with ampicillin.

On incubation overnight I saw colonies - picked them - conducted plasmid isolation. - then ran a small volume in gel but cant see any plasmids.

Now am lost.


You mean that you directly used the colonies on the plate and subjected them for plasmid purification? If so, I suspect that the plasmids in such a small colony is sufficent enough to be seen in the gel. You need to inoculate them into LB (+antibiotics) to culture them. Spin down the cell, and subject them to plasmid isolation. For high copy number plasmids such as pUC series, 1 ml of overnight cell culture contains several ug of plasmid DNA.

-zhongmindai-

If the pcr gives you one band don't need to gel extract (gel extraction give too low yield and if need sticky ends you will loose the polyA tails). If need to purify the amplicon use a spin column protocol. I used to add the amplicon to the vector with out any purification and got no problem.

What method do you use for plasmid extraction?? if using column maybe the column clogs and thast why don't have plasmid. If using the clasic method check the buffers, reagents and that the centrifugations are as stablish. The reagents have certain density to make sure that can separate the plasmid dna from the gdna. Load in the gel at least 6-10┬ÁL of samples.

-merlav-

How many colonies did u have?
I was told that you should have a good number of blue/white colonies, which then, most of the white would be positive (i know this is illogical, but i had the same problem with u the last time). <30colonies was really no good

wat I did was to make a few ratios of vector:insert ratio, pick all of them, and subsequently always use the ratio that gave the best yield.

-sharonpek-