problem with pWATERGATE plasmid

[ismawanto]

Currently we are working with pWATERGATE plasmid (18050 bp). We have transform this plasmid into ccdB survival E. coli (Invitrogen) by using heat-shock method, then spread it on LB plate containing 10 microgram/ml Spectinomycin. We trasferred the colony into LB media containing 10 microgram/ml. When we isolated the plasmid from E. coli culture, we got low concentration of plasmid. We use alkaline lysis with SDS method (Sambrook and Russell).
In order to confirm this plasmid, we digested this plasmid by using SacI and XhoI enzymes (double digestion). According to the restriction map, this plasmid only has one restriction site for those two enzymes. So after digestion it would produce 2 bands (16.3 kb and 1.7 kb). But for our case, after digestion it only produced single band (about 9.5 kb). Does it mean that we have isolated the incorrect plasmid.
Thank you in advance.

[Linda Zamariola]

Hello,
I'm also working now with pWATERGATE and I got the same problem you had. I digested the plasmid with Sac I and StuI (two fragments are expected after digestion) and I also made a digestion with Hind III, that is supposed to cut in three points. But, also in my case, the digestion produced only one single band of about the same size you had, 9,5 kb.
I'm also doubting if this is the correct plasmid. Did you manage to have results?
Thanks in advance.

[clevermizo]

Originally Posted by ismawanto

Currently we are working with pWATERGATE plasmid (18050 bp). We have transform this plasmid into ccdB survival E. coli (Invitrogen) by using heat-shock method, then spread it on LB plate containing 10 microgram/ml Spectinomycin. We trasferred the colony into LB media containing 10 microgram/ml. When we isolated the plasmid from E. coli culture, we got low concentration of plasmid. We use alkaline lysis with SDS method (Sambrook and Russell).

pWATERGATE is a Gateway system vector (Invitrogen). All of those plasmids are low-copy so you will get low yield (<100 ng/uL in a miniprep) unless you grow it in a larger batch and purify it from more culture. For low-copy plasmids and mini-preps, I usually start with 10 mL to 15 mL culture, but a midi-prep would be better.

In order to confirm this plasmid, we digested this plasmid by using SacI and XhoI enzymes (double digestion). According to the restriction map, this plasmid only has one restriction site for those two enzymes. So after digestion it would produce 2 bands (16.3 kb and 1.7 kb). But for our case, after digestion it only produced single band (about 9.5 kb). Does it mean that we have isolated the incorrect plasmid.
Thank you in advance.

What size does the undigested plasmid run?

This is a hairpin vector for RNAi. Typically RNAi vectors are prone to recombine, especially I imagine huge bulky ones like this, so you can't keep them in culture too long. How long do you grow your culture before you perform the plasmid prep (I would recommend <18 hours if possible)? Have you tried screening several different colonies after transformation or do you only subculture one colony? Have you tried inoculating culture directly from a glycerol stock?

[ismawanto]

Originally Posted by Linda Zamariola

Hello,
I'm also working now with pWATERGATE and I got the same problem you had. I digested the plasmid with Sac I and StuI (two fragments are expected after digestion) and I also made a digestion with Hind III, that is supposed to cut in three points. But, also in my case, the digestion produced only one single band of about the same size you had, 9,5 kb.
I'm also doubting if this is the correct plasmid. Did you manage to have results?
Thanks in advance.

I have tried many times. Until now I haven't got any positive result.

[ismawanto]

Originally Posted by clevermizo

pWATERGATE is a Gateway system vector (Invitrogen). All of those plasmids are low-copy so you will get low yield (<100 ng/uL in a miniprep) unless you grow it in a larger batch and purify it from more culture. For low-copy plasmids and mini-preps, I usually start with 10 mL to 15 mL culture, but a midi-prep would be better.

Usually I subculture one colony of pWATERGATE transformant into 5 ml LB media.

What size does the undigested plasmid run?

When I run the undigested plasmid, I got two bands. The first band is bigger than 21 kb and the second one (the more intense band) is about 9 kb.


This is a hairpin vector for RNAi. Typically RNAi vectors are prone to recombine, especially I imagine huge bulky ones like this, so you can't keep them in culture too long. How long do you grow your culture before you perform the plasmid prep (I would recommend <18 hours if possible)? Have you tried screening several different colonies after transformation or do you only subculture one colony? Have you tried inoculating culture directly from a glycerol stock?

Usually I subculture at least 10 colonies. I grow my culture overnight and then directly isolate the plasmid.
I don't have any glycerol stock. The company sent the plasmid on paper.

[ismawanto]

Originally Posted by clevermizo

pWATERGATE is a Gateway system vector (Invitrogen). All of those plasmids are low-copy so you will get low yield (<100 ng/uL in a miniprep) unless you grow it in a larger batch and purify it from more culture. For low-copy plasmids and mini-preps, I usually start with 10 mL to 15 mL culture, but a midi-prep would be better.

Usually I subculture it into 5 ml culture media.

What size does the undigested plasmid run?

When I ran the undigested plasmid on the gel, I got two bands. The size of the first band is > 21 kb and the second one (the more intense band) is about 9 kb.

This is a hairpin vector for RNAi. Typically RNAi vectors are prone to recombine, especially I imagine huge bulky ones like this, so you can't keep them in culture too long. How long do you grow your culture before you perform the plasmid prep (I would recommend <18 hours if possible)? Have you tried screening several different colonies after transformation or do you only subculture one colony? Have you tried inoculating culture directly from a glycerol stock?

For screening I subculture at least 10 colonies per plate. After subculturing, I incubate the culture overnight, then I directly isolate the plasmid.

I don't have any glycerol stock. The company sent the plasmid on paper.