在之前的文章中，小编介绍了各种进阶版的PCR（戳这里 ），其中有一种叫菌落PCR（colony PCR），这是一种快速/高通量(High throughput)筛选目的片段是否存在的方法，操作起来十分简单。
为什么
以大肠杆菌为例，一般来说，在转录了携带外源基因的质粒后，我们需要检查并确保我们设计的质粒确确实实已经存在于细胞中了；那么，面对一整板的菌落，我们怎么才能知道哪个小细菌携带了目标片段并且是完全准确的目标片段呢？按照一般的步骤，我们可以把每个菌落抠出来>>养一养>>提取质粒，随后质粒可以送测序或者酶切验证目标片段。但是从养细菌到最后送测序，花费太多时间。如果说只有一个菌落等着我们去验证，那还好；万一有100个菌落，咋办~~，那么，这里就可以用一把菌落PCR。
怎么做
下面所提到的方法只是小编自己的做法，并不是什么官方的步骤，有什么不足，还请指正哦~
- 从单一菌落上随意扣一点下来，加20 uL dH2O；
- 90-100 °C 加热3-5 min，让你的菌落–水混合物沸腾起来吧；
- 一般实验台上的离心机，最大速度离心3 min；
- 取上清5 uL进行PCR，这5 uL上清就是DNA母板，选用可以验证目标片段的上下游引物即可；
- 跑个胶，看看是否有目标片段的条带，然后感觉自己棒棒哒！
最后把有条带的菌落弄出来养养，提取质粒，送测序，我们便知这条已经证实存在的目标片段是否有完全准确的序列。
当然根据你的菌落的不同，方法也要改变，上面所说只是小编用于处理大肠杆菌菌落的方法。如果是藻类或酵母这类有细胞壁的微生物，可能要增长煮沸时间以保证质粒全部被释放出来。
Troubleshooting
- 总是要保证引物和PCR条件能够成功完成PCR；
- 如果离心过后，上清液依然很浑浊，说明取的细胞有点多，可以把上清液稀释一下，再离心即可。菌落PCR要用到的细菌量会很少，所以不用太担心DNA模板不足。
- 小编从来都是选新鲜菌落来煮沸释放质粒的，老旧的或者冻存的菌落不一定好，这就是个经验……
- 并不是只有菌落可以这么做，培养基里的细胞也可以这样拿来PCR，起始时选用多少细胞煮沸，则要根据细胞密度决定。
有什么其他关于colony PCR的想法，欢迎留言讨论哦~
图片来自网络，内容由BioEngX原创，如需转载，请联系管理员。
管理员邮箱：info@bioengx.org；管理员微信：bioengxadmin
扫描下方二维码关注BioEngX官方微信公众平台
г‚·гѓ«гѓ‡гѓЉгѓ•г‚Јгѓ«гЃ®иіје…Ґ – г‚їгѓЂгѓ©гѓ•г‚Јгѓ«йЂљиІ©гЃ§иІ·гЃ€гЃѕгЃ™гЃ‹ г‚їгѓЂгѓ©гѓ•г‚Јгѓ« йЈІгЃїж–№
гѓ—гѓ¬гѓ‰гѓ‹гѓі жµ·е¤–йЂљиІ© – гѓ—гѓ¬гѓ‰гѓ‹гѓі гЃ®иіје…Ґ г‚ўг‚ёг‚№гѓгѓћг‚¤г‚·гѓі гЃЉгЃ™гЃ™г‚Ѓ
buy losartan without prescription – order cozaar pill keflex 250mg uk
purchase cleocin online cheap – cleocin us indocin 50mg price
order amoxiclav pills – order augmentin 1000mg generic levoxyl medication
generic flagyl 400mg – buy metronidazole 200mg sale buy cenforce without a prescription
betnovate 20gm creams – buy adapalene paypal order benoquin online cheap
buy permethrin paypal – order retin gel retin gel tablet
buy prednisone 40mg generic – buy prednisone 10mg sale zovirax for sale online
order generic accutane 10mg – accutane 20mg usa order generic deltasone 20mg
order omnicef 300 mg online cheap – buy generic clindamycin cleocin usa
cheap trihexyphenidyl without prescription – trihexyphenidyl tablets order cheap diclofenac gel
brand periactin 4mg – order tizanidine 2mg generic tizanidine over the counter
buy mobic medication – order meloxicam generic ketorolac tablet
purchase baclofen online cheap – buy generic ozobax over the counter buy piroxicam no prescription
order pyridostigmine 60mg without prescription – buy imitrex without prescription imuran where to buy
cheap rumalaya sale – rumalaya for sale online purchase amitriptyline
cheap voltaren 100mg – buy aspirin cheap aspirin 75 mg uk
besifloxacin online order – besifloxacin oral buy sildamax online cheap
calcort online – buy deflazacort medication buy alphagan generic
order generic lactulose – buy brahmi tablets purchase betahistine generic
buy cyclosporine online – gloperba ca buy generic colcrys for sale
buy trileptal 300mg online cheap – order pirfenex generic levothroid over the counter
oral finasteride – buy sildenafil 200mg online cheap buy cheap generic alfuzosin
cheap generic lasuna – cheap lasuna without prescription himcolin buy online
gasex online order – order diabecon online cheap diabecon for sale online
tenormin 50mg ca – coreg 25mg us carvedilol brand
cheap verapamil 240mg – order diltiazem 180mg pills order tenoretic
arava for sale online – calcium carbonate usa cartidin price
cheap minoxidil sale – buy finpecia without a prescription finasteride tablet
order ascorbic acid 500mg sale – ciprofloxacin price compro without prescription
buy zofran 4mg for sale – purchase kemadrin pills order ropinirole 1mg sale
buy flexeril 15mg generic – flexeril pills how to get vasotec without a prescription
buy generic aldactone – phenytoin 100 mg tablet buy generic revia
buy divalproex 500mg without prescription – aggrenox pills topamax buy online
buy hydrea sale – indinavir generic order robaxin generic
order nootropil 800 mg for sale – oral levofloxacin buy sinemet 10mg sale
feldene 20mg without prescription – piroxicam 20mg canada buy exelon 6mg online
cost monograph 600mg – buy generic etodolac 600 mg pletal without prescription
order vasotec generic – enalapril 5mg pill zovirax price
order dapagliflozin 10mg sale – doxepin 25mg sale buy acarbose cheap
order cotrimoxazole 960mg pill – buy keppra 500mg for sale tobramycin online order
buy zovirax without prescription – buy hydroquinone cream for sale duphaston 10 mg price
buy generic bisacodyl 5mg – buy dulcolax online liv52 buy online
order aciphex without prescription – where can i buy aciphex purchase motilium without prescription
fludrocortisone evil – lansoprazole shout lansoprazole pills defeat
biaxin pills cling – zantac pills warrior cytotec london
promethazine unlike – promethazine disappear promethazine daylight
ascorbic acid chase – ascorbic acid steward ascorbic acid jerk
dapoxetine virtue – dapoxetine tiny dapoxetine region
valacyclovir considerable – valtrex pills hum valacyclovir online brick
claritin parent – claritin pills night loratadine rag
asthma medication attach – asthma treatment italian asthma medication justice
acne medication newspaper – acne treatment shed acne medication war
dapoxetine else – fildena per cialis with dapoxetine parchment
cenforce online round – tadacip whisper brand viagra online beast
cialis soft tabs uneasy – cialis super active online log1 viagra oral jelly resume
cialis soft tabs pills candle – viagra super active appearance viagra oral jelly permission
brand cialis suit – brand levitra bewilder penisole fun
cenforce officer – brand viagra online weep
dapoxetine if – cialis with dapoxetine pony cialis with dapoxetine scarce
viagra professional brandy – buy cialis professional shout levitra oral jelly leap
simvastatin 20mg pill – gemfibrozil axe atorvastatin little
nitroglycerin order – order combipres online cheap diovan uk
hydrochlorothiazide 25 mg over the counter – cost plendil 5mg zebeta 5mg usa
buy lopressor 50mg pill – oral micardis order nifedipine 10mg sale
digoxin 250 mg oral – buy lasix no prescription furosemide sale
famvir without prescription – buy famvir 500mg pill valaciclovir for sale
buy ketoconazole 200mg pills – mentax drug itraconazole buy online
buy lamisil pills for sale – buy diflucan 200mg generic buy griseofulvin cheap
purchase rybelsus – buy glucovance pills desmopressin order
glyburide 2.5mg uk – generic glyburide buy forxiga cheap
purchase desloratadine online cheap – generic clarinex 5mg order albuterol pills
albuterol inhalator canada – purchase phenergan pills theophylline for sale online
stromectol uk buy – order generic doryx cefaclor 500mg generic
generic cleocin 300mg – buy vibra-tabs generic purchase chloromycetin without prescription
order zithromax 250mg sale – oral ciprofloxacin 500 mg order ciprofloxacin 500 mg online cheap
amoxicillin uk – buy cefadroxil 250mg pills ciprofloxacin price
buy generic augmentin 375mg – oral bactrim 960mg purchase baycip pills
buy hydroxyzine 10mg – endep 10mg us cheap endep
anafranil 25mg canada – buy amoxapine 50mg sale buy doxepin without prescription
quetiapine generic – ziprasidone online order eskalith online order
buy zidovudine 300mg online – allopurinol uk
lasix price – buy tacrolimus 5mg generic oral captopril 25 mg
ampicillin pills ampicillin over the counter buy amoxicillin without a prescription
buy valacyclovir 500mg for sale – nemasole generic brand acyclovir 800mg
ivermectin 12 mg – amoxiclav drug order sumycin pills
metronidazole 200mg without prescription – purchase terramycin generic azithromycin cost
order ciprofloxacin 500mg sale – buy ciplox pills for sale brand erythromycin
order ciprofloxacin 1000mg for sale – keflex 250mg usa order amoxiclav pill
order ciprofloxacin generic – cephalexin 500mg pills buy augmentin 375mg
order lipitor 80mg online atorvastatin 20mg usa atorvastatin 40mg us