strange subcloning problem - (Feb/01/2012 )

Hi

I have a strange problem in my ligation

We ordered a synthetic gene from Genscript which is supplied in puc57 vector. I subclone the gene from puc57 into our self-constructed expression vector using NgoMIV and BsRGI sites. Below are our procedure

1. digest the insert and vector with BsrGI and then NgoMIV
2. gel purify the insert (~800bp) and vector (~10kb)
3. ligation in 1:3 or 1:5 ratio at 4C overnight or 16C for 4 hours
4. purify the DNA and then transformed in DH10b cells purchased from invitrogen

I usually get ~50 colonies in the ligation while my control without insert has ~0 to 10 colonies only.
however when i miniprep and check my plasmid, most of them are background from my insert prep, which is the puc57 vector with my synthetic gene in it.

i repeated it for many times but still no positive clones, I want some suggestion of it!

Maybe the digest is not complete or is not working properly.

if you can afford it, get Genscript to clone it for you into your vector.

You may be able to find an additonal enzyme which cuts your insert containing vector, but not your insert. If so, then cut with that enzyme in addition to the ones you need to release the insert. This will help reduce your background. This is all a lot easier if the vector you are moving to has a different antibiotic resistance.

Thx

I have tried to cut with an additional enzyme ScaI which cut at the ampicillin resistant gene of the insert containing vector but the background still appear.
i really run out of idea now

Please explain in ridiculous detail how you are cutting your vector and insert. Often problems arise from too high a concentration of DNA or enzyme in the reaction. There may be a reason for the poor cutting efficiency.

I cut ~0.5ug of my vector and insert in 40ul reaction vol using 10units of NgoMIV and BsrGI for ~4 hours. After running gel i could clearly see my insert ~800bp and vector ~9kb.
In the vector digestion i can also see a ~800bp fragment being removed, so I guess the digestion should be ok.

Then i purify my gel fragment and use ~10ng of vector for ligation using T4 ligase from invitrogen. I ususally use 1:3 ratio, or sometime I also try 1:5 1:1 1:10 etc


But after transformation and check clone, most of them come from the insert background

Not enough detail. What volume is your 500 ng of vector in (that is, what fraction of your 40 ul is DNA)? I thought you were also cutting with ScaI? Have you tried ligation without gel purification? Often gel UV exposure causes problems.

the concentration of my vector is ~150ug/ml, so i ususally cut ~3 to 4ul of DNA in total of 40ul reaction volume. i tried to cut with ScaI but since that time the result is no different i havent tried this again.

my insert is cut from a 3kb vector so i think i must need to do gel purification to separate my insert form its vector?

Well, given your constraints, I would cut the vector with NgoMIV and BsrGI in buffer 4. I would cut the Insert vector with NgoMIV, BsrGI, and ScaI-HF (The HF version works well in other buffers) in buffer 2. I would heat kill both reactions. I would CIP treat the vector reaction briefly with a minimal amount of CIP. This is a problematic step, and may be best simply avoided, but you may want to try several dilutions of CIP and several lengths of CIP treatment. You need to purify the CIP away, and it can't be heat killed, so a column cleanup or ethanol precipitation may work. This is why you want SAP or antarctic phosphatase. Then, I would mix them and ligate with normal ligation buffer in equimolar amounts (probably just equal amounts if they start with similar amounts of DNA in the digestions). I would not purify, but directly transform cells, recover for 1 hour at 37, and plate on selective media. You should have low background.

Hi miclee0710!!!

We are exactly having the same troubles with our GenScrip constructs build in pUC57. No matter the strategy we use, and although we are trying to digest as much as possible the pUC, the background always appears.

Were you able to finally solve the problem?

Thanks!