Molecular Biology/Cloning protocols

Below are brief in-house protocols for common molecular biology

procedures. For further information or updates, see the appropriate mfrs
instructions (Qiagen), or our molecular biology protocols tets.
!a"ing #B$%!& &lates
Bacterial transformation
!odified 'ransformation
Bacterial &lasmid (ulture
!ini &rep
&lasmid !idiprep
)*% )igestion
+rganic etraction$precipitation of )*% with &hase-loc" tubes (,enn
'aylor)
-el .ecipe
&(. of &lasmids
,enn 'aylor/s !olecular (loning 0uic" protocols
Making LB/AMP Plates
1. Find a 2essel with suitable 2olume (i.e. a 2essel that will hold
the entire 2olume of #B$%!& you plan to ma"e &#34 %'
#5%4' an 5Q3%# 6+#3!5 +F %7. to the amount of
li0uid in said 2essel).
8. 'o ma"e +*5 #7'5. +F #B$%!&, put 9:g of #B agar into
a 2essel with +*5 #7'5. of !illiQ water (9:g;1# water).
9. 'a"e a clean (washed in !illiQ) stir bar and place into the
2essel. 4tir on stir plate for appro. :min.
<. 'a"e 2essel (containing #B agar and stir bar) to the autocla2e
room and autocla2e for 9= minutes on the #7Q37)
45''7*-. *+'5; %utocla2e 2essel in a pan of water.
:. %fter autocla2ing, place a thermometer into the water pan
and allow to sit.
>. ?hen temperature is >=(, place 2essel with #B agar onto the
stir plate and begin to stir.
@. %dd 1==mg of %!&7(7#7* to the #B agar and stir for 8
minutes.
A. Q37(B#C pipet 9=ml of #B$%!& into &etri dishes. +ne
liter #B$%!& should ma"e appro 99 plates. *+'5; 4%65
'D5 B%- 'D5 &#%'54 (%!5 7* F+. 4'+.%-5 +F
&#%'54 7* 'D5 .5F.7-5.%'+..
E. %llow plates to cool on bench top o2ernight.
'he net day, !%.B 5%(D &#%'5 with +*5 B#%(B
4'.7&5, place the #B$%!& plates bac" into their bags, and
place in the refrigerator. *+'5; &#5%45 ?.7'5; #B$%!&
&#%'54 on the bag.
Bacterial transformation Protocol Using Heat
Shock
&urpose; 'o transform bacterial cells to ta"e up the plasmid$foreign )*%, so
amplify the )*%. 'he )*% is then purified and used in transfections, (=-7&/s, etc.
1) 'a"e out competent )Ds cells from FA= degree (elsius freeGer (:= ul
ali0uots) and thaw on ice for : minutes.
8) %dd 8 u# of circular )*% into cells and gently stir with tip. 7ncubate on ice for
9= min to thaw competent cells.
9) &ut tube(s) with )*% into water bath at <8 degrees (elsius for 8= seconds.
<) &ut tubes bac" on ice for 8 minutes to reduce damage to the cells.
:). %dd 1 ml of 9@ degree warmed'B (with no antibiotic added). 7ncubate tubes
for 1 hour at 9@ degrees (elsius.
>). 4pread about 1==-1:= ul of the resulting culture on warmed #B plates (with the
appropriate antibiotic added F usually %mpicillin or Banamycin). -row o2ernight
at 9@ degrees.
@) &ic" the colonies (8 per plasmid) about 18-1> hours later.
A) 'he colonies pic"ed for each )*% plasmid is placed in := m# #B and :-:= u#
of ampillicin, and then incubated in the 9@ degree (elsius sha"er o2ernight.
E) &urify )*% with midi prep.
Modified Transformation
&urpose; 'o isolate and$or increase the growth of a bacterial )*% plasmid for
use in transfections, (o-7&/s, etc.
1. 7f a plate is not made, ma"e plate accordingly; 9: mg agarose, 1 litre of dD
8
+, and 8
m# of ampicillan (:= mg$m#).
8. 4teriliGe the inoculating loop with @=H 5t+D and then flame. )ip into the eisting
plasmid stoc"$collection and strea" the plate; left, top center, right, bottom center, and
the middle.
9. &lace in the 9@ degree (elsius incubator o2ernight.
<. &ic" a large, but fairly isolated colony, and place in := m# of #B media and := u# of
ampicillan (plasmid bacterial culture protocol). &lace in 9@ degree incubator sha"er for
1< to 1> hours$o2ernight. 'his should greatly increase the )*% concentration yield.
Bacterial Plasmid Culture
&urpose; 'he purpose of this procedure is to grow$culture bacteria that contain a
specific )*% plasmid, which will be used in other eperiments, such as transfections and
immunoprecipations (7&/s).
1. +btain an autocla2ed 8:= m# erylmyer flas" and fill with := m# of #B broth, :=
ug$u# of ampicillen, and := u# of the bacterial plasmid.
8. 4wirl gently to mi and then place in a 9@ degree (elsius incubator$sha"er o2er night
(approimately 1>-1A hours depending on the growth of the bacterium.)
Mini Prep
&urpose; 'o isolate )*% from a bacterial culture and prep$purify it for )*%
digestions, gel identification, etc. Qiagen miniprep
1. Dar2est bacteria from culture tubes into 1.: m# centrifuge tubes; may ta"e 9-< spins
to har2est the entire culture from the tube.
8. &lace in the -8= freeGer for 1:-8= minutes (this helps lyse the cells better).
9. %dd <== u# of chilled complete eppendorf lysis solution, 2orte tubes briefly to mi,
and then incubate for 9 minutes at .'.
<. 'ransfer the solution to spin column tubes and centrifuge for 1 minute. )iscard flow
through and add <== u# of diluted wash buffer.
:. (entrifuge for 1 minute, discard flow through, and centrifuge again for 1-1.: minutes.
>. 'ransfer spin columns to collection tubes, add <= u# of filtered dD
8
+ and centrifuge
for one minute. .emo2e the column and store prepped )*% at -8= degrees (elsius until
ready for use.
Plasmid Midiprep
&urpose; 'o purify a plasmid of )*% from a bacterial culture, which will be
used in (o-7&/s and )*% identification gels and transfections. (Qiagen midiprep)
50uipment and directions are usually in the "it, but in case they are not, this is the
protocol that wor"s.
&repare a culture;
1. 'a"e a 1<ml (3#'3.5 '3B5, and fill it with 8ml 'B ('errific Broth)
8. 3sing a pipet tip, select an isolated colony from your plate by stabbing the tip
into the colony.
9. &lace tip into the 8ml 'B and -5*'#C swirl around.
<. %dd appropriate 2olume of the appropriate selection antibiotic to each tube (5I;
8ul of :=mg$ml %!& to 8ul 'B)
:. 'a"e tube to D5%'5) (9@() 4D%B5., place inside, and let sha"e for > hours.
>. %fter > hours, remo2e tube from sha"er.
@. 'a"e 8:ul of culture from the tube and place it into a flas" containing 8:ml 'B.
A. %dd appropriate amount of antibiotic (1ul of :=mg$ml %!& ; 1ml broth JK 8:ul
%!& :=mg$ml)
E. 'a"e flas" to D5%'5) (9@() 4D%B5., place inside, and allow to sha"e until
the net day.
1. %fter the bacterial cultures are at decent growth, ta"e :== u# of bacterial growth and
:== u# of glycerol and freeGe in -A=.
8. ?ith the rest of the bacterial culture, pour all of the := m# into a := m# conical
centrifuge and spin down for 8A minutes at >=== rpm in the cold centrifuge.
9. )ecant supernatant and resuspend the bacterial culture in : m# of chilled resuspension
solution.
<. *et add : m# of lysis buffer, in2ert > times, and let it incubate for 9-: minutes.
)uring the incubation, pull the plug from the syringe.
:. 'hen add : m# of chilled *eutraliGation solution to the lysis buffer, in2erting < times.
'hen add < m# of binding solution, in2ert twice and transfer to the syringe.
>. ?hile the solution is incubating in the syringe for about 9-: minutes, add < m# of
column prep solution to the empty column and let it 2acuum through.
@. Begin the push the solution in the syringe onto the column, careful not to o2erflow,
and 2acuum the solution completely through.
A. ?ash the column with < m# of wash 1 and then < m# of wash 8 solution. #et the
column dry for 8= minutes after the washes.
E. 'ransfer the column to a 1: m# conical centrifuge tube and spin down for : minutes
at <==== rpm. 'ransfer the column to a fresh collection tube and add 1 ml of elutant
solution or filtered deioniGed water. 4pin down for : minutes at <==== rpm. 'ransfer the
elutant to properly labeled 8.= m# centrifuge tubes.
1=. %dd =.1 2olume of 9 ! 4odium %cetate and =.@ 2olume of isopropanol, mi well
and then incubate for 8= minutes.
11. (entrifuge for 1: minutes in the < degree (elsius centrifuge at ma speed, remo2e
supernatant and add 1 m# @=H ethanol to wash )*% pellet.
18. (entrifuge for : minutes at ma speed, remo2e supernatant, and add <= u# deioniGed
water.
19. 'a"e the )*% and 0uantify it with the nanodrop and dilute )*% samples to desired
concentration; =.: ug$u# to 1.= ug$u#.
!A igestion
&urpose; 'o isolate a specific fragment of )*% from a bacterial cultureL
1. +btain a buffer that wor"s for all of the enGymes being used in the digestion (rac" <-1).
6orte well and "eep on ice.
8. 4et-up a master mi consisting the buffer (1,8,9, or <) that wor"s for all enGymes being
used in eperiment, filtered dD
8
+, and the enGymes. !ote" #eep the en$ymes in the
cold %lock and add last to the master mi&'
1= Buffer J 1 u#$1= u#, so 8 u#
5nGymes J =.8: u# for each enGyme used
?ater J add water until total of master mi sans )*% is e0ual to 1: u#
8. 5ach tube recei2es : u# of )*% and 1: u# of the master mi for a total of 8= u#.
'riterate the )*% and master mi well to miL ma"e sure there are no bubbles and use a
new tip with e2erything.
9. 7ncubate )*% digestion for 1-8 hours in 9@ degree (elsius water bath.
<. .un on a gel for 1-8 hours with a ladder$loading dye mi in a 1;8= u# dilution, and the
samples$loading dye in a 8=$< dilution.
(rganic e&traction/precipitation of !A )ith Phase*lock tu%es
+,enn Taylor-
1. Before the purification, obtain '?+ phase-loc" tubes (5ppendorff) for each
sample you want to purify, and spin them down in a micro-centrifuge for 9=sec at
!%I 4&55) (19,===rpm) at .'.
8. F+. (5## #C4%'5$)irty )*%; %dd up to 1ml of sample to the phase loc"
tube.
9. %dd an e0ual 2olume of phenol$chloroform solution (<() to the tube.
<. (5*'.7F3-5 in a micro-centrifuge at !%I 4&55) for 9.: minutes at .'.
:. %fter centrifugation, remo2e the top a0ueous layer from the phase loc" tube and
transfer to a *5? Mphase loc" tube. *+'5; )+ *+' '+3(D 'D5 -5#
#%C5. of the sample with the pipet tip. 7' ?7## (#+-.
%dd another 2olume of (hloroform (e0ual to that of the starting 2olume of
sample) to the new tube.
>. (5*'.7F3-5 in a micro-centrifuge at !%I 4&55) for 9.: minutes at .'.
@. %fter centrifugation, remo2e the top a0ueous layer from the phase loc"
tube and transfer to a 1.:ml epi tube.
@. !easure the 2olume of each sample, and add; 1$1= 2olume 9! 4odium
%cetate (<() %*) 8.: 2olumes 1==H ethanol to each.
&lace the samples at -A=( for %' #5%4' 8= minutes. *+'5; +nce at -A=(,
the samples can be stored indefinitely until you are ready to finish the
purification.
A. %fter -A=(, remo2e the samples and allow them to thaw at .' for :-1= minutes
(until (+!&#5'5#C 'D%?5)).
E. ?hen thawed, spin the samples at !%I 4&&5) for 9= minutes at <(.
1=. %spirate the supernatant from each sample ((%.5F3# *+#' '+ )74'3.B
)*% &5##5'), and wash pellet with A=H ethanol.
11. 4pin samples for 1= minutes at !%I 4&&5) at <(
18. %spirate the supernatant from each sample ((%.5F3# *+#' '+ )74'3.B
)*% &5##5'), and allow pellet to (+!&#5'#5C ).C in open air.
19. .e-suspend pellet in *F %utocla2ed water. *+'5; 7f pellet has trouble re-
suspending, heat sample to <=( for 9=sec-1min.
1<. Cou can now 0uantify )*% on nano-drop. 4tore at -8=(
.el /ecipe
&urpose; 'he purpose of this protocol is to set-up a gel for &(. of plasmid )*%,
.*%, ?estern blot, etc.
1. 7n an erlenmeyer flas", mi approimately A: to E: mg of agarose in 4B 1 buffer and
swirl gently.
8. 4tuff the top of the erlenmeyer flas" with a paper towel and heat the agarose miture
in the microwa2e for about two minutes in order to dissol2e the agarose.
9. #et the agarose miture cool to where you comfortably hold it without a o2en mitt and
add 1u#$1== m# of agarose gel miture, and swirl gently to mi.
<. &our into a gel electrophoresis compartment with the appropriate amount of wells
established for the eperiment and let it dry for <: minutes to an hour.
PC/ of Plasmids
&urpose; 'he purpose of this procedure is to amplify the plasmid )*% segment
and then run it on a gel for either identification, as a control, etc.
1. !i 1= u# of phusion DF buffer, 1 u# of 1= m! d*'&, appropriate amount of
plasmid )*% (the appropriate concentration; may ha2e to dilute with dD
8
+ to obtain
appropriate concentration) in a &(. tube.
8. %dd =.: u# of each primer being used in the &(. (usually 8 primers).
9. %dd dD
8
+ to the mi until it e0uals to <E.: u#. #astly add the =.: u# of the phusion
polyL Nadd phusion poly last to mi&0 %ecause it )ill %egin to denature !A once it is
added0 thus to ha1e the appropriate !A segment0 add last'2
2!(T3" !A mi&ture needs to add up to 45 uL0 so 1olumes of )ater and !A are
ad6usted to fit this criteria'
<. &lace the &(. tube in the &(. and set the amount of miture present and the amount
of cycles to doL the amount of cycles depends on amount of )*% needed for eperiment.
:. .un the amplified )*% on a gel.
22 Alkaline Phosphatase Treatment 22
4eongOin 4eo
1. %dd =.8unit of al"aline phosphatase (calf or shrimpL .oche 1unit$ul) for 1=g
)*%.
8. 7ncubate for 1= min at 9@(. )o not incubate longer than 1= min.
9. %dd 1$8: 2olume of =.:! 5)'% and heat-inacti2ate for 1: min at @=(.
<. )o agarose gel purification or &(. purification (Qiagen).
,enn Taylor7s %rief 1ersions of molecular cloning protocols
4teps for cloning;
1. &(. desired insert
8. .un &(. product on gel
9. 'a"e picture of gel. 5cise band of desired siGe
<. -el 5traction "it. 5lute in 9= ul water (Qiagen)
:. )igest 2ector and insert with desired enGymes o2ernight or : hrs at 9@ degrees
>. &(. purify insert. 5lute in 9= ul water (Qiagen "it)
@. .un digested 2ector on gel
A. 'a"e picture and 5cise lineariGed 2ector
E. -el purify ecised band. 5lute in 9= ul water (Qiagen "it)
1=. 4pec digested, purified insert and 2ector
11. (alculate 2olumes for ligation
18. #igate o2ernight at 1> degrees. %lso set up a ligation of digested 2ector alone
19. 'ransform ligations into 5F1alpha cells
1<. (ount colonies on plates
1:. &ic" colonies from 2ector P insert plate to grow as 9 ml 'B P %mp (:= ug$ml)
cultures
1>. 4igma mini preps of cultures (4igma "it)
1@. )iagnostic digests of mini preps (with same enGymes used to cut 2ector and insert)
1A. .un diagnostic digest on gelL loo" for insert to be cut out
1E. 'ransform correct mini preps
8=. grow cultures for midi preps (8: ml or := ml o2ernight culturesL 2arious "its to use)
81. 4e0uence to chec" (send to &*%(#)
%lternati2ely (subcloning);
1. )igest a plasmid containing the insert and the desired 2ector with the same or
compatible enGymes o2ernight or for : hrs at 9@ degrees
8. .un products on a gel
9. 'a"e a picture and ecise desired bands
<. -el purify. 5lute in 9= ul water (Qiagen "it)
:. #igate o2ernight at 1> degrees. %lso set up a ligation of desired 2ector alone.
Follow steps 19-81 abo2e
&ossible alternati2es;
1. 7f two enGymes do not cut in the same buffer, digest in one enGyme for : hrs or
o2ernight. &(. purify the digest (Qiagen "itL resuspend in 9= ul water). )igest with the
second enGyme for : hrs or o2ernight. .un 2ectors out on gel to ecise band or do a
second &(. purification for inserts.
8. (an (7& digested 2ector, but 7 usually don/t
9. +ther diagnostics are colony &(. or to run out undigested mini prep compared to the
2ector alone if the insert is greater than 1 "b.
<. For digests. )*% should be less than the total 2olume. 5nGymes should total less than
1=H of the total 2olume. For 2ectors or plasmids, 7 digest A ug in 1== ul. 1=I buffer and
1=I B4% should be diluted 1;1= final.
(7&/ing a 2ector
1. dilute 1=I al" phos buffer to 1I in :=ul total 2olume
8. remo2e 1 ul, add 1 ul of al" phos enGyme (=.=83$ul)
9. set up reaction ; 8== ul total 2olume
1== ul digested )*%
8= ul 1=I al" phos buffer
for e2ery 1.> ug plasmid, add 1 ul of al" phos enGyme in 1I al" phos buffer
<. 7ncubate in 9@ degree D8+ bath for 9= minutes
:. (lem up by running o2er &(. prep column
>. 5lute in := ul 5B
@. .un lineariGed 2ector on gel to remo2e uncut portion
A. -el purify (Qiagen) and elue in 9= ul 5B
E. Quantitate
&(. purification (Qiagen)
1. %dd : 2olumes of Buffer &B to 1 2olume of &(. reaction. !i.
8. %pply samples to Q7%0uic" columns. (entrifuge for 9=->= seconds
9. )iscard flow-through. .eplace Q7%0uic" column into same tube
<. %dd =.@: ml Buffer &5 to Q7%0uic" column. (entrifuge for 9=->= seconds
:. )iscard flow-through. &lace Q7%0uic" column bac" in same tube. (entrifuge for an
additional minute.
>. &lace Q7%0uic" column in a clean 1.: ml microfuge tube
@. 'o elute )*%, add := ul or 9= ul Buffer 5B (1=m! 'ris-(l, pD A:.) or dD8=
A. #et stand 1 minutes
E. (entrifuge 1 minute.
Qiagen -el 5traction
1. 5cise )*% fragment from agarose gel with a clean scalpel or raGor blade
8. ?eigh gel slice. %dd 9 2olumes of Buffer Q- to 1 2olume of gle (1== mg Q 1== ul)
9. 7ncubate at := degrees for 1= minutes (or until get slice is dissol2ed). !i by
2orteing tube e2ery 8-9 minutes during incubation.
<. (hec" that color of mitures is yellow (7f color is orange or 2iolet, add 1= ul 9 !
sodium acetate, pD :.= and mi.
:. %dd 1 gel 2olume of isopropanol to sample and mi
>. %pply sample to Q7%0uic" column (<== mg per column) and centrifuge for 1 min.
@. )iscard flow-through and place Q7%0uic" column bac" in same tube
A. %dd =.: ml Buffer Q- to Q7%0uic" column and centrifuge for 1 min.
E. %dd =.@: ml Buffer &5 to Q7%0uic" column and centrifuge for 1 min.
1=. )iscard the flow-through and centrifuge the Q7%0uic" column for an additional 1
min.
11. &lace Q7%0uic" column into a clean 1.: ml microfuge tube
18. 'o elute )*%, and := ul or 9= ul Buffer 5B (1= m! 'ris-(l, pD A.:) or D8+ to
column
19. #et stand 1 minute
1<. (entrifuge for 1 minute
#igations;
!i;
?ater; to 1= ul
1=I '< ligase buffer; 1 ul
1=m! %'&; 1 ul
1=m! !g(l8; 1 ul
2ector; 1 ul
insert; 1-< ul
'< ligase; 1 ul
&lace at 1> degrees o$n
For (alculations of eact amounts of 2ector and insert;
6ector; 8=-1:= ng
6ector$insert lengths J R
)i2ide ng of 2ector in 1 ul by the 2alue of R J Q
!ultiple the 2alue Q by the ratio of insert to 2ector desired (for eample 9 to 1) J .
)i2ide . by the concentration of insert to obtain the 2olume of insert to use
(olony &(.
Qiagen;
1=I &(. buffer; 8.: ul
1=m! d*'& mi =.: ul
:/ primer (=.: ug$ul) =.: ul
9/ primer (=.: ug$ul) =.: ul
'a0 =.18: ul
dD8+ to 8: ul
Blenta0;
1=I buffer : ul
d*'&s 1 ul
:.8 ! betaine 18.: ul
Blenta0 =.1 ul
dD8+ to := ul
&ut mi in &(. tubes
'ouch tip to colony, to master plate to mi. #et tips sit in mi for a few minutes
(apped m.*% (m!essage m!achine "it)
#ineariGe 1: ug plasmid
&henol$choloroform and chloroform etractions
&recipitate with glycogen, 9! *a+%c, and 5t+D
.esuspend in 1= ul dD8+
'ranscription;
1. 2orte 1=I reaction buffer. &lace at room temp.
2orte 8I *'&$(ap mi. &lace on ice
8. !i at room temp;
water; : ul
linear )*%; 1 ul
1=I reaction buffer; 8 ul
8I *'&$(ap; 1= ul
enGyme mi; 8 ul
9. &ipette up and down
<. &lace at 9@ degrees for 8 hours
:. %dd 1 ul )*ase 7. !i well. 7ncubate in 9@ degree bath for 1: min.
>. %dd 11: ul *uclease-free water and 1: ul %mmonium acetate stop solution
@. 5tract with; 1:1 ul phenol$chloroform and then 1:1 ul chloroform
A. %dd glycogen, 9 ! *a+%c, 1==H 5t+D. &lace at -A=
E. 4pin 9= min at < degrees
1=. ?ash with A=H 5t+D (1 ml) 8I
11. .esuspend the pellet in 1: ul .*F dD8+
18. .un 1 ul on =.AH agarose gel.
Calculating molar ratios for ligations
1. Decide how much vector will be used in ligation (i.e. 20ng)
2. Figure out how much larger the vector is than the insert
Example: ou have 0.!"b insert and a #.#"b vector
#.#"b$ 1! (since vector is 1!% larger than insert& ou need 1!% less
0.!"b insert)
'. Figure out how man ng o( insert ou will need (or a 1:' vector:insert ratio
Example: 20ng (amt o( vector ou are using in ligation) $ 1.'ng o( insert (or 1:1
1!% ratio& so multipl b ' to get 1:' ratio)
B%($&%( prep
1. start a := ml culture o2ernight with antibiotic
8. har2est bacteria by centrifuging at >=== for 1: min.
9. resuspend the bacteria in 8.< mls of '54 (1=m! 'ris pD @.:, 1 m! 5)'%, =.1!
*a(l). %dd <.A ul of := mg$ml #ysoGyme (1== ug$ml final) 7ncubate at room
temperature for : minutes
<. %dd 8.< mls of =.8 ! *a+D$1H 4)4 (ma"e fresh daily). !i gently by
in2ersion. &lace on ice for : minutes. 4hould become clear as cells lyse
:. %dd 8.< mls of 9! B%c ('o >= mls of : ! B%c, add 11.: mls of glacial acetic
acid and 8A.: mls of water) !i by in2ersion. &lace on ice for : minutes
>. (entrifuge at 1=,===g for 1= minutes.
@. 'ransfer supernatant into a polypropylene centrifuge tube
A. %dd : ul of 1= mg$ml )*ase free .*ase %. 7ncubate at 9@ degrees for 1 hour.
E. add an e0ual 2olume of phenol$chloroform (1;1). !i by in2ersion. 4pin at
98:=g for 1: minutes
1=. 'ransfer a0ueous phase to a culture tube. %dd an e0ual 2olume of isopropyl
alcohol. !i by in2ersion. 4pin at 1=,===g for 8= minutes.
11. resuspend in 8== ul dD8+. 'ransfer to a 1.> ml epi.
18. %dd 1$1= 2olume of 9 ! *a+%c (pD :.8) and 8.: 2ol 1==H 5t+D
19. 4tore at -A= degrees for S hour to o2ernight
1<. 4pin in centrifuge at < degrees for 9= minutes at top speed
1:. wash pellet with 1 ml of A=H 5t+D
1>. 4pin for : minutes at < degrees at top speed
1@. .esuspend in := ul buffer 5B ('ris)
%d2antage 8 &(.
!i;
?ater; 1A.: ul
1=I &(. buffer; 8.: ul
d*'& mi; 8 ul
'emplate; =.: ul
:=I &olymerase; =.: ul
F primer (=.: ug$ul); =.: ul
. primer (=.: ug$ul); =.: ul
&rogram; 'ouchdo, 'ochdown
1. E< degrees for 1;==
8. E< degrees for =;1:
9. >A degrees for >;==
<. goto 8 <I
:. E< degrees for =;1:
> :: to >A degrees for =;9=
@. >A degrees for >;==
A. goto : 8EI
E. @=.= degrees for 1=;==
1=. <.= degrees fore2er
'ransformation;
1. 'haw )D:alpha cells on ice (1I or less for ligationsL 1I or 8I for plasmids)
8. !i 1 ul plasmid or 8-9 ul ligation with 8: ul )D: alpha cells
9. 7ncubate on ice :-9= minutes (7 usually do 1= minutes)
<. Deat shoc" at 9@ degrees for 9= sec (D8+ bath)
:. 7ncubate on ice 1-8 minutes
>. %dd 1@: ul 'B and reco2er at 9@ degrees sha"ing for 9= (plasmids, %mp) or >=
(ligations, Ban) minutes
@. &late onto #B P %mp (Ban) (@:-1== ul)
A. grow o2ernight at 9@ degrees