Discover the world's scientific knowledge
With 135+ million publication pages, 20+ million researchers and 1+ million questions, this is where everyone can access science
You can use AND, OR, NOT, "" and () to specify your search.
Hi, My colleague is trying to do the LR reaction to move an artificial miRNA from the entry vector to the destination vector. Both vectors have the same antibiotic resistance gene. He has tried:
1) Linearize the entry vector and setup LR reaction with destination
vector ( pGWB2)
2) Linearize both entry and destination vector before LR reaction
3) incubated over night
4) Linearized destination vector using the recommended restriction
enzyme and then set up LR reaction
Any help here would be greatly appreciated because we are stuck and we need to get this done very badly. Sarah
I know two ways: one is PCR followed by Taq polymerase reaction with dATP at 72°C, and another is PCR followed by RE digestion of PCR product having restriction sites in primers. Are both methods equally effective or is there a better way?
I cleaved a vector with two enzymes, after which I would like to blunt the ends and dephosphorylate before ligating to an insert. The insert was PCRed out from a different vector and then the ends were cleaved with an enzyme that creates blunt ends. I would like to reduce the purification steps to a minimal so that I can conserve the amount of DNA I have. Below is the process that I believe would be sufficient, please let me know if there are any additional purification steps that are required or if there are any I can skip:
Vector: Double digest -> gel purify -> Blunt the ends -> dephosphorylate -> ligate
(if I am using an alkaline phosphatase for the dephosphorylation I believe I can inactivate it and therefore skip the purification step before ligation?)
Insert: PCR -> gel purify -> restriction digest -> gel purify -> ligation
I just got one for five constructs, the other three can not be successful, and I can not find the problem. this method was reported as more than 90% positive. Is there anyone knowing why, please tell me, thank you!
I'm performing combined RNA isolation protocol: phase separation by TRIzol and chlorophorm and spin (silica) collumn one from adherent tissues. I was wondering if I can store cell pellets suspended in TRIzol and after collecting of whole group of samples thaw them and perform isolation of good quality RNA. Have you tried this approach?
I have been trying to clone a gene of slightly more than 4kb into pLVX vector using single blunt and sticky end ligation (PmaI/XbaI site on gene, SmaI/XbaI site on the vector).
I did ligation using T4 DNA ligase at 16C overnight, at various ratios (1:3, 1:4, 1:5, 1:6, 1:10) with 50ng vector in 20ul reaction.
Then I transform 100ul of competent Stbl3 cells using 10ul of the ligation mix as follow:
->put on ice 30mins after mix
-> heat shock 60 secs
-> put on ice 2min
-> added 300ul LB (w/o Amp) and recover at 37C for 1-2 hours
-> spin down cells at 4000rpm for 5 min
-> resuspend cells and spread on plate
My positive control plate with uncut vector (100ng, about 9kb) usually yield just around a hundred colonies and most of the time I get 0 colonies on the experimental plates.
I have been stuck at this for months. Not sure if it's problem with transformation or cell competency. I've tried making fresh competent cells and did get over few hundred colonies, but no colonies mostly for other plates. Except for once I get a single colony but that doesn't seems to contain my gene. Not sure whether the vector+ insert is too large (nearly 13kb) to be cloned? Any suggestions or advice?
I have a vector backbone of 5.5Kb in which I need to ligate an insert of ~600bp. The Roche T4 DNA ligase manual I'm using says ligation should be kept for 16h @ 16 deg. Celsius. My question is that does it really take that long or I can transform the ligation after 7-8h incubation at the said temperature. If not, can something be done to accelerate the ligation?
Note: I do not have the option of using fast ligases that are available commercially. I have only Roche T4 DNA Ligase, which recommends 16h incubation for successful ligation.
Thanks in advance...
I know high background can occur if plasmids are not properly digested, or if they self ligate. I understand longer incubation times and dephosphorylation of the vector can minimise background but are there any other ways?