Professional Documents
Culture Documents
Mini Prep
Mini Prep
GTC Lab 4
GTC Lab 4
9. Centrifuge for 30 60s. Discard the flow-through.
10. Wash the bound plasmid DNA by adding 0.75 mL of Buffer PE and
centrifuging for 30 60 s.
This step is necessary to remove salts from the plasmid DNA. Salts can inhibit
restriction endonuclease digestions.
11. Discard the flow-through, and centrifuge for an additional 1 min to remove
residual wash buffer.
Buffer PE contains ethanol. Thus, it is very important to remove any residual
buffer as ethanol will inhibit subsequent enzymatic reactions.
12. Place the column in a clean microcentrifuge tube. To elute DNA, add 50 L
Elution Buffer (EB) or water to the center of each column, let it stand
for 1 min, and then centrifuge for 1 min.
DNA is released from the silica-gel membrane by its low salt content. This
buffer is 10 mM Tris, pH 8.5.
Part 2: Recombinant plasmid analysis by restriction enzyme digestion
1. Set up your analytical restriction enzyme digestions for each plasmid.
Use ~1 g of DNA in each reaction. Assume the concentration of plasmid DNA in your
purifications is ~0.2 g/L.
Make a master mix for 5 reactions of all components except plasmid. Pipet the
appropriate amount into 4 microcentrifuge tubes leaving room to add plasmid.
x 5 rxns:
___ L
___ L
___ L of 10X BSA (some enzymes do not require this see buffer chart)
___ L
0.5 L of enzyme (of each enzyme if you are doing a double digest with two enzyme)
___ L sterile distilled MilliQ water (to 20 L total reaction volume)
20 L total volume
2.5 L
___ L
100 L
GTC Lab 4
2. Incubate the reactions at 37C for two hours. Add 2 L of 10X loading buffer to the digest
and load 20 L onto a 0.8% agarose gel containing EtBr (0.5 L). Electrophorese at 100 V for
45 minutes. Load 5 L of 1 kb ladder in one lane.
Part 3: Recombination into GFP vector
The L-R recombination reaction
The LR Clonase enzyme is a purified form of phage Lambda recombinase. It will remove the
coding sequence from the pENTR/D-TOPO vector and replace the gateway cassette in the new
pIGF-GTW vector for transformation into Tetrahymena. Recombining your cloned gene into
this new vector will create an in-frame fusion of your gene with GFP. Its expression will be
under the control of the MTT promoter, which is inducible with divalent metals such as
cadmium.
SettinguptheLRrecombinationreaction
LR Clonase II enzyme mix is supplied as a 5X solution.
1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
1-3 l of pENTR plasmid clone (50-150 ng total)
1 l of pIGF-GTW destination vector (400 ng/l)
H2O to 4 l
2. To each reaction above, add 1 l of LR ClonaseII enzyme and mix well by pipeting up
and down being careful not to introduce air bubbles.
3. Incubate reactions at room temperature overnight.
Your instructor will store them at -20C the next day.
GTC Lab 4
General
Part 1: Plasmid isolation
Aliquot the plasmid isolation buffers from the kit so that there are 3-4 sets available for
students to use.
Encourage students to mix their bacterial cultures well before taking 1.4 mLs. Many cells
will have settled to the bottom of the culture tubes.
It saves time if students come to lab prepared with knowing which enzymes they will
use for restriction digestion. The enzymes they use should cut once (or twice) in the
vector and once (or twice) in their cloned piece of DNA such that it will yield a banding
pattern allowing them to distinguish whether their gene was cloned, and in which
direction it was inserted. This is a good prelab assignment for them to figure out. A
restriction map of the pENTR vector is included. This may be copied and given to
5
GTC Lab 4
students in advance. They should already have restriction sites mapped on their gene
from the bioinformatics module.
If no restriction sites can be found for the available enzymes, digest with NotI (cuts
once in the vector) and students will be able to see if the shift in band size is consistent
with that expected for cloning the correct sequence. It will not yield information on
direction, but only a small fraction of inserts go in the wrong direction.
While the restriction digests are incubating, students should pour their agarose gels so that
they are ready to load as soon as the digest is complete.