ࡱ> \_[e Ubjbj .Xebeb&bF` j vvv8<dI&tbb(eee%%%%%%%$'s*x%9veeeee%vv&etvv%e%Vt#@8$@p# %&0I&#x*I*8$*v8$Xeeeeeee%%LeeeI&eeee*eeeeeeeeeB &: Basic cloning A basic cloning reaction involves putting one piece of DNA (the insert) into a plasmid backbone (also called a vector) that can be replicated in E. coli. The insert DNA is usually obtained by PCR. In the simplest cloning, PCR primers are designed that will amplify a piece of DNA and add restriction enzyme sites to each end. This insert is digested with appropriate restriction enzymes, as is the plasmid backbone, and the two pieces of DNA are ligated together to give the desired product. What restriction enzymes should I use? There are a huge number of restriction enzymes available, but it is best to stick to the common ones as they tend to work better. For a standard cloning you should consider: The chosen enzymes must not cut within the insert The chosen enzymes must cut the plasmid backbone only at the insertion point The enzymes should cut 100% in the same buffer (see the NEB catalogue for this information). 75% is acceptable if there is no alternative. Be careful with NEB HiFi enzymes as these have different buffer preferences. The enzymes should not have compatible sticky ends Both enzymes (one at least) should cut to give sticky ends, not blunt ends Both enzymes should preferably be already in the lab (check the enzymes box) This is a list of good enzymes for standard cloning: BamHI BglII EcoRI HindIII KpnI NcoI NdeI NotI PstI SacI SalI SpeI SphI XhoI ClaI and XbaI are blocked by some combinations of Dam/Dcm methylation which means that they are complicated and best avoided if possible. Cloning steps PCR is used to create the DNA insert(s), with primers carrying restriction sites at the 5 end A plasmid backbone is obtained (usually by growing cells containing the plasmid from a frozen stock and extracting plasmid DNA) The PCR insert is digested and gel extracted The plasmid backbone is digested and purified by gel extraction The insert is ligated into the plasmid backbone The ligation is transformed, colonies grown up and plasmid DNA extracted Test digests are used to ensure that the PCR product has been successfully integrated in plasmid backbone The insert (if derived by PCR) is sequenced PCR to create insert Primer design: Design oligos to amplify your DNA of interest (see PCR protocol). Add restriction sites to the 5 ends, with 10bp of junk on 5 end. Restriction enzymes cut poorly at the ends of fragments, so the 10bp of junk dramatically improves digestion. Ie: your oligos should be of the form [JUNKJUNKJU][Restriction site][Gene specific sequence] Run a 50l Phusion and a 50l Q5 PCR reaction for each desired insert (you just need one to work, it doesnt matter which), protocols are in the PCR protocol. Run out 5(l PCR to check that there is a single band of the correct size, and purify the rest of the PCR using a QIAQuick PCR purification kit, elute in 34(l. Digestion of insert and backbone Add to the insert: 4l 10x restriction buffer 1l each restriction enzyme Incubate at 37( for 1 hour Meanwhile, digest the plasmid backbone: 500ng plasmid DNA in 34l total volume 4l 10x restriction buffer 1l each restriction enzyme Incubate at 37( for 1 hour Run the entire products of both digests on a 1% agarose gel containing SYBR Safe and extract the required bands under blue light not UV. Do NOT image the gel using the UV transilluminator prior to gel extraction this massively reduces efficiency Purify bands using a QIAQuick or NEB gel extraction kit, elute in 30(l Ligations Set up ligations as follows: water6 l-3 lvector2l2l-insert-6l-ladder--5(l Add 1(l 10x ligase buffer and 1(l T4 ligase (NEB), incubate 30min at RT or overnight at 16( If ligated vector will be larger than ~5kb, overnight at 16( can be necessary. Ligase buffer should be thawed at RT (not 37( - bad for the ATP and DTT) then vortexed until white precipitate (DTT) dissolves. If ligations fail it is normally the buffer not the ligase that is at fault. Visualise the ladder ligation on a 1% agarose gel. The ladder ligation should contain almost entirely high molecular weight species. If it still looks ladder-like, repeat the ligations with fresh ligase and buffer. Transform 5(l of the first two ligations in 50(l competent cells. We normally use Invitrogen subcloning grade DH5 cells, transformed as recommended Miniprep 2-6 colonies and perform test digests. Try to select restriction enzymes that will produce a clearly defined product that is different between the desired product and the plasmid backbone alone. Test digest: 2l DNA 1l 10x restriction enzyme buffer water to 9.6l 0.2l (2-4U) each enzyme Variations: Multi-fragment ligations You can easily ligate two inserts into the plasmid backbone at once if building a complex plasmid, just make sure that the sticky ends of the different fragments can only be combined in the desired way. We routinely do four fragment ligations, but these are a little less reliable. Cloning into a single restriction enzyme site It is sometimes necessary to use an insert with the same restriction site at either end to clone into a single site in the backbone plasmid. In this case, proceed as normal, but after digesting the plasmid backbone for 1 hour, add 1U rSAP and incubate at 37( for a further hour. Proceed to ligation as normal, but when screening colonies use restriction enzymes that will reveal the orientation of the insert. Ligation of oligo fragments A small insert can be made by annealing two oligos. The oligos should be designed so that when they are annealed they will leave sticky ends compatible with the cut plasmid backbone. Mix 45(l each 100pM/(l oligo (not phosphorylated) and 10(l 10x restriction enzyme buffer (any) heat to 95( and slow cool to RT Continue directly to the ligation without gel purification or any other cleaning step If ligating into a single site in the plasmid backbone, design your oligos so that in the ligated product the restriction site is destroyed. Do not CIP treat the plasmid backbone. Perform the ligation in an appropriate restriction enzyme buffer +1mM ATP and 0.25(l restriction enzyme, and after ligation heat inactivate the ligase and digest 1 hour at 37( with another 0.25(l restriction enzyme. Generating blunt ends This protocol will blunt one end of the DNA, in general clonings with one blunt end work well, clonings with all blunt ends are harder to control. Digest the DNA with the restriction enzyme that needs to be blunted, using a normal 50l digest as described earlier. Then chill the reaction to 12(, add 0.5l 10mM dNTPs and 0.33l T4 DNA polymerase, incubate for 15min at 12(. Add 1l 0.5M EDTA and incubate 20min at 75(. Clean the DNA through a QIAQuick column, elute in 44l. Add the buffer and restriction enzyme for the second digestion and proceed as normal. T vector cloning To add A overhang to PCR product: Mix 30(l purified PCR product 0.6(l dNTPs 3(l 10x ThermoPol buffer 0.3(l Taq (NEB) Incubate at 65( for 2 minutes To 1.5(l tailed PCR product add the following from T-Vector kit (Promega): 2.5(l 2x ligase buffer 0.5(l pGEM-Teasy 0.5(l Ligase Incubate at room temperature for 30 minutes (can reduce if desperate) Transform ligation into 50(l DH5( cells, remember to spread 40(l 20mg/ml X-Gal in DMF on each plate before spreading the transformed cells Tips for awkward clonings that refuse to work Dephosphorylate the vector If you have lots of colonies on the vector-only plate, you can dephosphorylate the vector prior to ligation, preventing re-ligation of the vector ends. This reduces efficiency across the board so high efficiency cells are best to transform after this: Mix 17l purified vector DNA 2ul 10x CutSmart 1l rSAP Incubate 30 min at 37 then 5 min 65 Use directly in ligation reactions Clone the insert PCR into a shuttle vector Restriction enzymes do not work brilliantly at the ends of DNA, so sub-cloning increases efficiency. You can either clone into a T-vector using the protocol given above, or (more quickly) use a CloneJET PCR cloning kit: Mix 1ul purified Phusion PCR (not restriction digested, preferably gel purified) 2.5l 2x buffer from kit 0.25l pJET plasmid 0.25l T4 DNA ligase 1l water 30 min at RT then transform and plate on AMP plates normally >50% colonies are positive Not all sticky ends are the same GC rich overhangs (eg: NotI) can be fully hybridised at RT when you set up ligations (which favours wrong product), whereas AT rich ones (eg: PacI) may not stably hybridise at all during an RT ligation. Do the following to help both problems: Mix your Vector, insert and water for ligations but no buffer or enzyme. Heat to 65 for 5 min then cool to RT 5 min on the bench and spin briefly. Add the buffer and ligase then ligate at 4 over night Use absent and incompatible enzymes Most enzymes work a bit in ligase buffer, so after ligation you can heat inactivate the ligase (65, 20 min) then add a restriction enzyme (37 1 hour) to remove unwanted products. This can help in two situations: You are cloning from a shuttle vector and you get the original vector instead of the product back (it happens). If there is a restriction site in the shuttle vector that is not present in the final ligated product, you can cut with that. If you design your primers so that the restriction sites are not the same ones as in your destination plasmid but are compatible (e : A $ K a   : B Q R ) *  ) + , / 1 3 6 : = ? A D J M O R U X [ Ľįѯįѫѧїяїїїїh1h"M 6h1hkn6h1h@)6h@)h"M hg hg5 hkn5 hknhknhz hkn6h*)hknhmhmh{5CJ(\aJ(hmhm5CJ(\aJ(hOYq5CJ(\aJ(7 # $  R * ] + , O r gd"M gdkn & Fgd"M $a$gdm[ ^ a d e g j m p r u x { ~ BCD|)BJMyz " ҽ㬤h& OJQJ^Jh& hTFh~?xh&h&h&5 hx+~5h*)h|hLz hkn5 h rh@) h r6h rh< h<6h1h"M 6h"M hknh1hkn6hhhhhh66}*jMy12STgdLz ^`gdLzgdg & Fgd*)  -./012TiUVabc{| h~?x5 jmhLzhg@ hIhLzjh~?x0J>*Uh)}hLz>* h)}hLz hLzhLz jhLz hLz5hLz jmh& hADhIh~?xh"M jmh&h& h& h& h& h& h& OJQJ^J. *Fbc\] $IfgdLzgdg ^`gdLzgdLz56AGOpq(*68<P^789;<GHc->|}~ԜhIhI5 hZ0hZ0hPWhZ0 hZ05 hhiMN hiMN5 h5hLzhLz5 hLzhLzhADh jhLzh~?x jmhLz jmhhhLzhLzhiMNhLz5 hLz5 hAD55^UUUU $IfgdLzkd$$Ifl\$    t0 644 laytLz^UUUU $IfgdLzkd$$Ifl\$    t0 644 laytLz  ^UUUU $IfgdLzkd$$Ifl\$    t0 644 laytLzrs^YYYPPGG^gdLz`gdLzgdLzkd$$Ifl\$    t0 644 laytLz 6789:;HIbc~gdIgdmgdggdLz^gdLzGHIde! $ % 2 3 V W w } !!!!!!!!Y"Z"l"m""""""##$$=$>$$$$$%%+%,%7%8%T%U%o% jmhh&h5 h5h jh h5hhcKT jh" jmh"h"hh"5 h5 hI5h jhIh[hI;HIef  """".#/#$$$$%% %&%4%O%gdgd"gd$gdgdmgdIO%a%%%%%%%&&N&O&&&& ' '(')'%(&(D(W(b(c((((gd"`gdgdo%p%%%%%%%%%&&i&j&o&p&&&&&&&& '(')'4'T'c(l(y((((((((())*L*O*P***,+K+++ ,º«h>g h& 6h& OJQJ^J h& h& h& hTFhTFOJQJ^JhTFh& hTFhTFhTFhTF6 hTF6 hTF5hTFhTF5 h& 5 hkdh jah h&h jmhh jh2(((())*"*7*M*X*Y*****++,,a,b,,,,,--. & Fgd>ggd" ,!,h,,,,,,,,!-#-M-N-/TTTT\U]U^U`UaUcUdUfUgUiUjUtUuUUUUUUUUUUUUUUUԥsjhLz0JU*h[0JmHnHu* hLz0JjhLz0JUhLzmHnHuhLzjhLzUjhUhh~?xjh~?x0JU h>gh>gUhTF h>g6 h& h& h>gOJQJ^Jh>gh& h& OJQJ^J-g: SalI and XhoI), you can digest the ligation product with SalI and XhoI and only the desired ligation product will survive (because ligation of a SalI fragment to an XhoI fragment produces the sequence GTCGAG which is not a site for either enzyme).  In our hands, using blue light for gel extraction not UV is THE thing that makes cloning work!      FILENAME Cloning.doc v2.2 Houseley lab  PAGE 5 .T\U]U_U`UbUcUeUfUhUiUUUUUUU & Fgd>g,1h. A!"#$% $$If!vh#v #v#v#v:V l t0 65 555/ / / / /  / / ytLz$$If!vh#v #v#v#v:V l t0 65 555/ / / / / / / ytLz$$If!vh#v #v#v#v:V l t0 65 555/ / / / / / / ytLz$$If!vh#v #v#v#v:V l t0 65 555/ /  / / / /  / ytLzs2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRi@R  Table Normal4 l4a (k (No List jj zB Table Grid7:V04@4 x+~Header  9r 4 @4 x+~Footer  9r .)@!. x+~ Page Number>@2> @) Footnote TextCJaJ<A< @)Footnote Text Char@&`Q@ @)Footnote ReferenceH*PK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< &ad&X 88EEEH[ o% ,U$')O%(.U !"#%&(+ #9@BH!8@0(  B S  ?,1AHJNOSUYaegkmqrvx|~    r z ;A>G]a* . a!c!e!i!% % %%%%D%H%M%Q%%%%%&e&g&h&j&k&m&n&p&q&&& &e&g&h&j&k&m&n&p&q&&&333::~~ { &e&e&h&h&q&&&&&&::~~ { &&i6e[G{ft-.^`o() ^`hH. pL^p`LhH. @ ^@ `hH. ^`hH. L^`LhH. ^`hH. ^`hH. PL^P`LhH.h^`OJQJo(hHh^`OJQJ^Jo(hHohp^p`OJQJo(hHh@ ^@ `OJQJo(hHh^`OJQJ^Jo(hHoh^`OJQJo(hHh^`OJQJo(hHh^`OJQJ^Jo(hHohP^P`OJQJo(hHh^`OJQJo(hHh^`OJQJ^Jo(hHohp^p`OJQJo(hHh@ ^@ `OJQJo(hHh^`OJQJ^Jo(hHoh^`OJQJo(hHh^`OJQJo(hHh^`OJQJ^Jo(hHohP^P`OJQJo(hH{fe[i6                           PO-m"M & o c[ h& N! a&@)016V58;Hq;<A?g@ZwAzBTFiMN TcKTPWC_kdaye>grnOYq7t~?x{|n|)}1~x+~"=g!sWLz*)P Ah ep tZ0z roNIsM 1kn'M;g$xADI&&&@HHHH4%&@8@@UnknownG.[x Times New Roman5Symbol3. .[x Arial?= .Cx Courier New;WingdingsA$BCambria Math"1h֛G EW DW D!4%% 2Q HP ? 2!xx& Basic subcloningHouseleyBaptiste Piguet   Oh+'0p   , 8 DPX`hBasic subcloning HouseleyNormalBaptiste Piguet12Microsoft Office Word@ޡ @.k@tW  ՜.+,0  hp  Edinburgh UniveristyD% Basic subcloning Title  !"#$%&'()*+,./012346789:;<=>?@ABCDEFGHIJLMNOPQRTUVWXYZ]^aRoot Entry FK`@Data -1Table5+WordDocument .XSummaryInformation(KDocumentSummaryInformation8SMsoDataStore@0AWSUCJBCVWH==2@Item  PropertiesUCompObj r   F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q