Help w/ CLONING...lots of transformants but no insert... - (Jan/02/2007 )

Hi all,
I am having some problem with my cloning...I RE (BamH1 and EcoR1) an insert from pCDNA3.1 plasmid and ligate it with BglII and EcoR1 digested pDsRed2N1 vector... ligate the sample ON at 4C and did transformation...usually I have lots of colonies 30-300 on my LB-Kan plate but so far none of the clone I screen carry my plasmid....
Since my insert is only a 162 bp it's clear to see it after running the pCDNA3.1 w/ insert on gel--> gel extraction
for my pDsRED2N1 RE vector I have tried cutting it for 2.5 hours, 4 hours, and Overnight....(just in case the vector wasn't completely digested...)--> gel extraction)
did a 1:5 ligation...but still have no clone w/ insert in it?
the way I screen my clones is by RE them w/ enzyme sites that are not present if there is ligation.. (used Xho1 and HindIII)


any tips? thanks in advance

Hi... ,

have you tried to cut the vector pDsRED2N1 with each RE itself or do you do an double-digestion?
It sounds as if you would perform a religation and outcompete your insert in the ligation.
It's difficult to say what is wrong but make your your pDsRED2N1 is cut properly.
Do you perform a religation control? If not you might do that as well; it tells you if this is the problem in your ligation!

I would guess it's your pDsRED2N1 digestion and therefore and or religation afterwards.

Good luck,
Cheers

It sounds like one of the enzymes (Bgl II or EcoR I) you are digesting the vector with is not 100% effective. This can be circumvented by treating the vector with SAP so that linearized vector cannot religate. Alternatively, since the Bgl II site is destroyed when you ligate the insert, you can try cuttnig the ligation products with Bgl II before transforming. This will lineraize any plasmids resulting from incomplete EcoR I cuts.

how does ur negative control plate look like (the one with the vector + ligase but no insert).

And if there were no colonies in this plate but u had the 30-300 colonies in ur ligation plate, I would advise to use a lot of miniprep DNA for RD to confirm its presence.

Thx Guys...I will try all the sugguestions and see if I can finally get it..


Bomber...I did a double digestion instead of single, but I got my other clones (other insert) into pDsRed vector using double digestion w/ BglII/HindIII and it work... but I will start doing single digestion just in case thx

tfitzwater... if it's incomplete digestion...can I just do a double digestion and let the reaction run overnight, say 16 hours, instead of 4...then everything should be digested right?

Scolix... i didn't do a negative control plate ... but I have sequenced my plasmid (pDsRed and pCDNA3.1 w/ insert).... would u suggest that I still do a negative control? I just determine that the clone has no insert because "Xho1 or HindIII" can cut my clone...these sites shouldn't be present if there is insert (RE Sites of pDsRed: BglII---XhoI--HindIII-EcoRI) ... is that a okay way for screening or?

Thx all

...yes, always do a negative control (a religation control of your vector).
Cut it as usual, perform a ligation without an insert and see if you get colonies after transformation. If no, your problem is not religation and/or unsuffecient digestion with RE I would say... .

Good luck.

U must have a negative control to confirm that ur vector is completely digested and that the colonies u c r not religation of the vector.

I am wondering if the u had some other site upstream of xhoI which u could use to digest the miniprep DNA. I am wondering if something is preventing digestion of xhoI site.

Also as I suggested earlier, try using more DNA for digestion so that u could visualise the small fragment.

Good luck !!!

The optimal sodium concentrations for these restriction enzymes ranges from 50 mM to 150 mM NaCl. While double-digests are possible, they tend not to go to completion. In addition, at least two of the enzymes are prone to star activity. SAP can be used simultaneously with the restriction enzymes when digesting the vector.

I recommend KGB for double digests. 10x Potassium Glutamate Buffer (KGB) is a universal restriction buffer. ( M. McClelland et al. 1988 NAR 16 (1) 364. and J. Hanish and M. McClelland 1988 Gene Anal. Techn. 5: 105-107) The buffer consists of 1 M potassium glutamate, 0.25 M Tris acetate, pH 7.6, 0.1 M magnesium acetate, 5 mM b-mercaptoethanol and 500 µg/mL BSA. Combine 3.33 mL of 3 M potassium glutamate, 625 µL of 4 M Tris acetate, pH 7.6, 1 mL of 1 M magnesium acetate, 3.5 µL of 14.3 M b-mercaptoethanol, 500 µL of 10 mg/mL BSA and 4.54 mL of Type I water. Prepare 1 mL aliquots and store at -20°C. A salt gradient will occasionally form if the tube is frozen. Mix well before use. A working tube may be kept at 4°C for over 1 year. Several commercial versions are available (REact 9 from Invitrogen, Y/Tango from Fermentas, New England Biolab's Buffer 4 and GE Healthcare’s One-Phor-All-Plus). Some enzymes requiring high salt, such as Sal I, may require addtion of NaCl to 100 mM final.

Overnight digests carry the risk of nibbling from the contaminating exonucleases that are typically present in most restriction enzymes.

The negative controls (vector only plus and minus ligase) should result in less than 50% of the colonies from the vector plus insert plus ligase. If you don't have at least twice as many colonies on the plus insert plates, most of the colonies will be minus insert.

Hi all, just to tell u guys that no colonies grow on my negative control plate and I have sent my pDsRed clone to sequencing...
but now i am running into trouble w/ another clone in pGEX4T-1 vector...I can get plasmid and can SEE it on my agarose gel after EasyPrep...but after screening and believe it's the clone w/ my insert i inoculate another tube of culture --> did miniprep/plasmid prep using commerical kit, but NO band is detected...the Nanodrop (spec) said i have ~340ng/ul DNA for my plasmid prep but nothing is being detected on agarose gel... i have repeated this with another pGEX4T-1 clone and it's the same.. nothing... but this kit works well for my other preps and well for others.... anyknow know what's wrong?

Plasmid DNA Easyprep (Adopted from BioTechniques 14(4):527-528 (1993)
　inoculate 2 mL medium with a single colony and grow overnight (12-18 h)
spin 1.5 mL (30 sec, full speed, microcentrifuge), keep rest in refrigerator
remove supernatant
resuspend pellet in 30 - 100 µL lysis buffer
boil the suspension for 60 sec and immediately chill on ice for another 60 sec
centrifuge at maximum speed for 15 - 20 min at RT
add RNase A (final concentration: 0.2 µg/µL) and incubate at 37 °C for 30 - 60 min