15 replies to this topic

[kartiga natarajan]

Dear friends....

                I could not succeed in doing mutation....I redesigned the primers and tried from the beginning but i could not get...Im getting colonies after transformation but it is not having plasmid...Please help me regarding this...I have attached the pictures of my results n the protocol i used....In transformation im using controls too... kindly go through n give ur suggestions...

Attached Files

•  Quick change mutagenesis .ppt   163.5K   26 downloads

[GNANA]

i think your PCR is working fine and i dnt think your Dpn digestion doing any harm here...you saying you didnt get plasmid, is it your procedure or the kit nor working?  

Make sure your competent cells are good, in terms of sterility and efficiency?


besides this, what i would suggest is after Dpn digestion run the whole product, do a gel extraction, elute in 10 microlitre sterile water and transform.  i usually transform half of the eluted volume or sometimes even the whole depending on the PCR yield and the efficiency of my competent cells.

Note: when u run the whole product after dpn digestion, sometimes u ll see lik a smear around the actual product, dnt panic lookin at that, jus cut around and do a gel extraction.

[kartiga natarajan]

Thanks Gnana for your helping hand...I screened colonies doing plasmid isolation manually but im getting plasmid from the positive plate. Also im sure my competent cells are good since im getting good results in positive control. As per your advice i will try doing gel extraction and get back to you. Thanks....

[kartiga natarajan]

Gnana...As per ur suggestion i tried gel extraction and transformed but again i got only one colony similar to the previous...all my transformation controls work...Can u plz help me to sort the problem...Where might be wrong?

[GNANA]

how much plasmid you used for ctrl transformation and how many colonies you got in that? your competent cells commercial or lab made?

[kartiga natarajan]

I transformed 1.5ug concentration of  plasmid in control and i have got more than 1000 colonies from it. The competent cells im using are lab made by Cacl2 Mgcl2 protocol

[kartiga natarajan]

Also after Dpn1 digestion i did not get any smear around the product as you said...I really dont know where will be the problem? plz do help me in sorting this Gnana...

[GNANA]

Do you mean 1500 ng (1.5 microgram) of ctrl plasmid and got over 1000 colonies? if this is the case your cells are way down the required efficiency, which should be over 10^9 cfu/microgram plasmid.  For mutageneis transformation i would say your cells shd atleast give 10^7 cfu per microgram. so, you got to prepare good cells and then proceed instead of repeating in the cells you already have.

About Dpn smear just dnt mind, even i dnt always visualize, here we cannot blame the digestion; if the problem is with inefficiecnt Dpn, you would still get colonies but wit wild-type plasmid.

if you want to ensure your competent cell efficiency, tranform some 10 pg of plasmid and count the colonies.

[kartiga natarajan]

Is there any nice protocol to prepare this efficient competent cells other than Rbcl...Since in our lab it is not available....Can u give me a nice protocol?

[GNANA]

you can find enough information to make good competent cells here  http://openwetware.o...competent_cells

[kartiga natarajan]

Hi Gnana...As per ur suggestions i prepared Top10 competent cells and i got colonies in both DH5alpha and XL1 blue competent cells....I also screened for the plasmids and the plasmids are also fine....further im also trying to verify using Restriction digestion and PCR...So give me an idea how many plasmids shall i send for sequencing? both from XL1 and DH5alpha? Thanks in advance

[GNANA]

To start with i would sequence 3 plasmids. mostly i ll get all three positive for mutation,  then i choose 1 or 2 from it and sequence the entire insert to make sure there is no other PCR induced variations.

[kartiga natarajan]

Gnana my gene was cloned in Xho1 & EcoR1 sites....So i took some plasmids from XL1 & DH5alpha transformed after following mutation protocol....But i could see out of them only one plasmid has released the insert...My positive control also have worked....What would be the reason? Shall i give the ones which have released the inserts after digestion 4 sequencing? I am also doing PCR with those plasmids...Kindly give me your valuable suggestions....

[GNANA]

Actually there shd'nt be a probelm with the presence of insert,  but make sure the improper digestion is'nt the cause for that. anyways you also checking them with PCR. personally i prefer restriction digestion  than PCR, as the possibility of false positivies/negatives are more with PCR screening.  however, with proper controls you can interpret that.

your positive controls that worked for digestion is a similar prep (miniprep/maxi or plasmid isolation protocol) like the mutated preps??

sometimes PCR induced variations may abolish the RE, but i believe you would hav used high fidelity taq or PCR mix that could be used for mutagenesis.

if not the above, make sure your cells are sterile.  some times you ll get colonies due to contaminaton... but following transformation procedure without adding plasmid or just water could help you to check the sterility of the cells.

since you are having the insert issues in the fellow preps,  double check the plasmids that released the insert are specific by digesting with other RE's, and then i think you can send them for sequencing, dnt forget to sequence a wild-type plasmid along.

[kartiga natarajan]

No the positive control is the  maxi prep plasmid isolated from kit...But i screened the plasmids manually...Also i used Pfu Turbo DNA polymerase only...While isolating plasmids manually i did not give 70 percent ethanol wash...Will that be the reason?Im sure my competent cells are sterile...While running plasmids after isolation all plasmids showed same retardion compared to my empty vector. Will take your suggestions Gnana....Thanks