REs at MolBi

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About restriction enzymes (REs).

At MolBi, we are currently making the transition to "Fast Digest" restriction enzymes from Thermoscientific (formerly Fermentas). The reason is, that Invitrogen (for years our major supplier) appears no longer to be a reliable supplier of REs.

From April, 2011 we started buying all REs from one-and-the-same supplier, which is Thermoscientific.
All enzymes we use are currently available as FD (“FastDigest”) enzymes from this company.

In daily use, FD enzymes are 10-20% more expensive than regular enzymes, but have several advantages..:

they cut very fast (may not be very important in most cases..),
but also: all FD enzymes cut in the same buffer (with or without Loading dye; both are supplied..), so double-digestions are no problem!

Enzyme concentration (units per ul) are not given anymore, but all enzymes are titrated so that one microliter digests one microgram DNA in less than 15 minutes.
Since the enzymes are more expensive, a series of test digestions were done at MolBi using only 0.5 ul: perfect result (checked after 30 minutes digestion)..
So, that will be the advice: use 0.5 ul for each digestion (pipet the viscous enzyme solution in the buffer/DNA mixture, and wash all enzym solution out by pipetting up-and-down). See also the exact reaction conditions info.
FD enzymes will appear more and more in the familiar -20 boxes.
10x FD buffer, and 10x FD Green (includes Loading Dye) can be found in the Fermentas Buffers -20 box.

Several users have noticed that using more than 1 ul per 20ul reaction mixture (e.g. when doing double or triple digests) gives incomplete digests. A double digest in 20ul using 0.5 plus 0.5 ul enzymes is OK; triple digests should be made in 30ul using 0.5 plus 0.5 plus 0.5 ul of enzymes.
The composition of the 10xFD buffer is not known, but it should be kept in ice, and stored in -20. It is probably also wise to store it in small aliquots to avoid multiple freeze/thaw cycles.

Unfortunately, this all means, that during the transition, we have five types of enzymes/buffers combinations:

Invitrogen/REact buffers (see REactBufferChart, REactBuffersComposition)
(in original Invitrogen vials, or in vials with hand-written label)
Invitrogen New /H, M, L Buffers (see NewBuffersComposition)
(in original Invitrogen vials, or in vials with hand-written label, both marked with a circled N on the label)
NEB/NEBuffers (see info)
(in original NEB vials, or with hand-written label marked with “NEB”)
Fermentas conventional/Fermentas buffers (see info)
(in original Fermentas vials, or with hand-written label marked with “Ferm”)
FD Fermentas/FD buffer (see info)
(in original FD vials, or with hand-written label marked with “FD”)

Currently, it can be hard to figure out which buffer is best for a particular double digest, but please look forward to the near future when we will use one buffer (FD Buffer) for all..

Fermentas is now a Thermo Scientific company (i.e. from 2012).

In the table below, you'll find all existing Restriction Enzymes. It is an excerpt from the Fermentas catalogue.

Use the table like in this example:

imagine, you need to digest your DNA using Eco47I. The table shows, that we do not have Eco47!, but the Eco47I table row shows, that we do have the same enzyme, but with a different name: AvaII.

Consider the price of an enzyme, especially when you need to digest many DNAs to check a particular construct! Use the FastDigest price as a guideline.
To find cheap enzymes, see this lists: FD enzymes pricelist, FD pricelist cheap to expensive (note: this list contains all FD enzymes available from ThermoScientific, not only enzymes present at MolBi (those are the ones in red in the table below).

You may be interested in an ApE enzymes file containing MolBi enzymes as a group.

Save money for the lab by using the very inexpensive miniprepping protocol when prepping a large number of clones..

Other usefull info:

About Taq polymerase,

PCR problems,

Inexpensive Miniprepping,

DNA size markers

A printable list of all Gateway vectoren/constructs at MolBi

3-point ligation: a useful trick in plasmid constructions..

Red names are available at MolBi , possibly as FD enzymes (last edited: maart 11, 2014)

About Taq DNA polymerase
LabZero is the supplier of our commonly used Taq DNA polymerase.
The batches from LabZero are made by overexpressing the protein in a bacterial clone. A standardised purification procedure is used, but batches may still vary in quantity and quality.
Before making it available, a new LabZero batch of Taq is always compared with earlier batches by the performance of a series of dilutions in a standard PCR, to determine the concentration in units/µl. LabZero produces once or twice a year a large batch of Taq DNA polymerase. Each batch has a new name. The stock is kept at -20°C as a very concentrated solution, and is diluted and dispensed in colored vials containing 200µl of 2 units/µl Taq. For most applications 0.5 unit (0.25 µl) per PCR reaction (50µl) will be sufficient.

The composition of 10x concentrated Taq buffer is:
100 mM Tris pH8.3 / 15 mM MgCl₂ / 500 mM KCl.

LabZero recommends this about the use of the Taq:

always wear gloves when handling the Taq working stocks tubes. Skin bits could contaminate the Taq with DNA!!
LabZero Taq preps are made using a rather quick-and-dirty protocol.., so, adding more Taq may result in worse results, due to the introduction of more contaminants!
in case of PCR products in the water control, please read the info below..

The special heat-stable, proof-reading Phusion DNA polymerases is also available at MolBi (for cloning purposes only!!).

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PCR product of the expected size, ALSO IN THE WATER CONTROL?
Think about it:
if you applied PCR products on an agarose gel
(maybe by mixing the PCR reaction mixtures, containing large amounts of DNA, with Loading Dye Solution by pipetting up-and-down, CAUSING AEROSOLS!!)
and use the same pipet for setting up the next PCR with primers that can use the PCR products you just loaded, as a template, in that case you may introduce template DNA into your master mix, giving all those problems....
Solve this problem, by leaving the lower parts of your pipet(s) for a weekend in about 1% Extran soap, followed by using tap water, ethanol and press-air to wash and dry them before assembling again.
Realise that solutions you have have worked with using the contaminated pipets may still contain minor amounts of your template DNA!
Try to use fresh solutions if you still have some..
(Unfortunately we cannot afford to use cotton-plugged pipet tips)

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About DNA size markers:
Available at MolBi is a ready-to-use DNA Ladder Mix and (sometimes) a 100bp DNA Ladder.

Or make your own, by digestion of Lambda DNA.
A few suggestions:


Lambda DNA Eco91I (BstEII) digest	LambdaDNA EcoRI+HindIII digest	Lambda DNA PstI digest

For 200 µl marker mixture, enough for about 20 gels, you could do the following:
Add to 105 µl [100 ul] of water:
20 µl of the appropriate 10x digestion buffer
70 µl of Lambda DNA (0.3 mg/ml)
5 µl (50 units) of the RE
[+5 µl (50 units) of a second enzyme].

Incubate for at least one hour at 37°C.
Add 25 µl of 10x DNA Gel Loading Solution, and run 10 µl of this mixture to get a nice marker lane...

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GeneRuler DNA Ladder Mix (from Fermentas Life Sciences):

note:
it has been reported, that this ladder may show a band pattern that is not consistent during a gel run!
For example:
After a 1.5 hours gel run, an expected 2950 bp plasmid fragment band may appear halfway the 3000 and the 3500 ladder bands, while 2 hours later the plasmid band clearly comigrates with the 3000 ladder band, as expected...

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GeneRuler 100bp DNA Ladder Plus (from Fermentas Life Sciences):

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Save money for the lab and try the protocols below, as an alternativ for the expensive miniprep kits from Sigma (€ 1.00 / column) or Omega BioTek (€ 0.78 / column) , when you only need to prep for subsequent digestions..

** Rapid Boiling prep according to our former collegue Andreas Untergasser.
(very easy, though not extremely quick, but clean enough for sequencing!!)

** Quick & dirty miniprep

(extremely easy, very quick; clean enough for RE digestions, not for sequencing..:
see steps 1 to 18 below)

In all steps, except step 15, supernatants can be removed (or transferred to a fresh epje) very fast by pouring (off), rather than using a pipet!

centrifuge (“spin”) 1.5 ml o/n bacterial culture: 20 seconds/max rpm

immediately pour off supernatans (‘sup”), but leave about 50 -100μl

resuspend cells in the remaining sup by vortexing
(should take 15-30 “)

add 300μl TENS (0.1M NaOH/0.5% SDS in TE)

mix by inverting about 5 times

add 150μl of 3M NaAc pH5.2

mix by inverting about 5 times

spin: 5 minutes/max rpm

pour the sup to a new 1.5 ml vial

add 900μl of absolute (or clean 96%) ethanol

mix by vortexing for a few secs

spin: 5 minutes/max rpm

pour off & discard sup

wash pellet with 500μl 70% ethanol
(= add 500μl 70% ethanol, and vortex for a few seconds;
pellet should still be intact)

take off all liquid
(= first pour off all sup; close the tubes and spin for a few seconds, and remove and discard all remaining sup using a yellow pipet tip)

be sure that no liquid is visible in the tube; leave tubes open for at least 10 minutes to evaporate all ethanol

dissolve the dried pellet in 30μl of water containing 20μg/ml RNaseA
(use the vortex; a milky suspension should easily be formed. At that point stop vortexing..)

Spin down the insoluble white stuff (2 minutes/max rpm) and use the clear supernatans in subsequent digestions

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latest update: maart 11, 2014
by JanV


Enzyme	Specificity 5’→3’	Price per ul	Fermentas' conventional enzyme name	Commercially available isoschizomers


AanI	TTA↓TAA		AanI (PsiI)
AarI	CACCTGC(4/8)↓		AarI
AasI	GACNNNN↓NNGTC		AasI (DrdI)	DrdI, DseDI
AatI	AGG↓CCT		Eco147I (StuI)	PceI, SseBI, StuI
AatII	GACGT↓C	1.27	AatII	[ZraIm]
Acc65I	G↓GTACC	1.27	Acc65I (Asp718I),[KpnIm](GGTAC↓C)	Asp718Im
AbsI	CC↓TCGAGG
Acc16I	TGC↓GCA	1.49	NsbI (FspI)	AviII, FspI
Acc36I	ACCTGC(4/8)↓		BveI (BspMI)	BfuAI, BspMI
AccB1I	G↓GYRCC		BshNI (BanI)	BanI, BspT107I
AccB7I	CCANNNN↓NTGG		Van91I (PflMI)	BasI, PflMI
AccBSI	CCGCTC(-3/-3)↓		MbiI (BsrBI)	BsrBI
AccI	GT↓MKAC		XmiI (AccI)	AccI, FblI
AccII	CG↓CG		Bsh1236I (BstUI)	BspFNI, BstFNI, BstUI, MvnI
AccIII	T↓CCGGA		Kpn2I (BspEI)m	Aor13HI, BseAIm, Bsp13I, BspEIm, MroIm
AciI	CCGC(-3/-1)↓	2.40	SsiI (AciI)	AciI, BspACI
AclI	AA↓CGTT		Psp1406I (AclI)	AclI
AclWI	GGATC(4/5)↓		BspPI (AlwI)	AlwI
AcoI	Y↓GGCCR		CfrI (EaeI)	EaeI
AcsI	R↓AATTY	0.86	XapI (ApoI)	ApoI
AcuI	CTGAAG(16/14)↓		Eco57I (AcuI)	AcuI
AcvI	CAC↓GTG	0.16	Eco72I (PmlI)	BbrPI, PmaCI, PmlI, PspCI
AcyI	GR↓CGYC		Hin1I (BsaHI)	BsaHI, BssNI, BstACI, Hsp92I
AdeI	CACNNN↓GTG		AdeI (DraIII)	DraIII
AfaI	GT↓AC	[0.41], 0.64	[Csp6I (CviQI)m](G↓TAC), RsaI	[CviQIm], [RsaNI]
AfeI	AGC↓GCT		Eco47III (AfeI)	AfeI, Aor51HI
AfiI	CCNNNNN↓NNGG		BseLI (BslI)	Bsc4I, BslI
AflII	C↓TTAAG	0.42	BspTI (AflII)	AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464I
AflIII	A↓CRYGT
AgeI	A↓CCGGT	3.72	BsaTI (AgeI)	AgeI, AsiGI, CspAIm, PinAI
AgsI	TTS↓AA
AhdI	GACNNN↓NNGTC		Eam1105I (AhdI)	AspEI, BmeRI, DriI, EclHKI
AhlI	A↓CTAGT		BcuI (SpeI)	SpeI
AjiI	CACGTC(-3/-3)↓		AjiI (BmgBI)	BmgBI, BtrI
AjnI	↓CCWGG	[0.18]	EcoRIIm , [MvaI (BstNI)](CC↓WGG)	[BseBI], [Bst2UI], [BstNI], [BstOI], Psp6I, PspGIm
AjuI	↓(6/11)CCAA(N)7TTC(12/7)↓		AjuI