cloning experimental design to ligate three PCR products into one plasmid - (Sep/26/2006 )
Hi, All,
this forum is indeed helpful for my cloning experiment! I found tons of useful info and tips here...
I am now planning another cloning experiment, trying to ligate three different PCR products into one target plasmid (see the attachment for the plasmid map, pEVS79).
My rough design is the following:
SacI--first product--NotI
NotI--2nd product--SpeI
SpeI--3rd product--KpnI.
I would like to ligte three PCR product at the sites I list here. Since I do not want to bother going to TA cloning vector in the first place, I am thinking of ligating PCR products directly (of course cut with proper enzymes before ligation)into the pEVS79. How does it sound? Of course, I can do the ligation one piece at a time.
But if I wanna ligate three pieces in the same tube with SacI-KpnI cut pEVS79, is it still feasible? Is there any special requirement for the ratio of these pieces during the setup? When I design the PCR primer, I know I should add extra bases in front of the restriction sites. But how many bases should I add? are 7-8 bases enough? what is the sequence of the extra bases? same for each enzyme or different enzymes have different requirement?
I would like to hear your comments on this design....thanks a lot!
cheers
Paula
I've had success with ligating two products and the vector in a single reaction, so I think your plan is feasible. I used a 1:1:1 molar ratio and 300 ng total DNA in a 20 ul ligation reaction. I concentrated the ligation mixture using a kit and used the entire amount for transformation. For your plan I would do controls for the vector alone and also combinations of two inserts and the vector.
For the primer design, just check the NEB catalog under "cleavage close to the end of DNA fragments". This shows the optimal number of base pairs for each enzyme and the recommended sequence.
A four way ligation is certainly feasible, especially as insertion into pEVS79’s MCS can be screened by X-gal/IPTG colour testing (lacZ disruption). However, you should be willing to screen a fair number of white colonies, up to 96 clones (which isn’t much with a multichannel pipette and colony PCR)
As for the ratios, I make sure that the insert fragments have a 1:1:1 mol ratio to each other. I stress mol, as the number of molecules of insert is important and not the number of grams. The insert:vector ratio I use something like 3:1 mol ratio. Ligate overnight at 16 Celsius.
As for the PCR primers, extra base pairs have to be inserted behind the restriction site
Random example
GCTAG GGATCC GTACTCTAGAAATA
Guard : GCTAG
Restriction site: GGATCC
Binding Sequence : GTACTCTAGAAATA
As for the number of bases to add to the guard, a good place to look at would be NEB website, its technical reference webpage. I usually use 5 bp, though for some enzymes, more bases must be added (8bp). As for the sequence of the guard, I use oligocalculator, to find a sequence which is relatively GC rich and causes minimal self complementary within the entire primers sequence..
hi, thanks so much for the tips and useful website link!
I browsed the NEB technical support website and I need to double check my understanding with you here:
SacI seems to be picky--even after 20hr of digestion, only 10% can be cleaved. And NotI need at least 8bases for cleavges. and it seems that all the enzymes I shall use (SacI, NotI, SpeI, Kpn) all need at least 20hr for digestion....
But will 20hour digestion generate star activity?
thanks...loosk forward to your reply!
cheers, Paula
But will 20hour digestion generate star activity?
SacI, with 1 bp overhang, won't cut well. Give it a longer overhang, I won't use anything less then 5bp, and it will be fine.
Yup, NotI needs at least 8bp of overhang.
As for the digest, NEB rates 20hr for 1 microgram of oligo DNA with 20U of enzyme.
Increasing the amount of enzyme in the digestion would speed up the digest. I use about 30U. The volume of enzyme should not excess 5% of the digest volume, as the glycerol which comes with the enzyme will inhibit the reaction.
Star activity during long digest is not major a concern with the common restriction enzymes. It has more to do with incorrect buffer conditions and excessive glycerol. Cap the tube tightly and cover the heating block to avoid condensation on the lids. (A thermal cyclers with the heated lit on (set at 37 Celsius) would do the same.