- Mohammad Ayoub added an answer:10Can anyone help me with DpnI-mediated site directed mutagenesis?
I am trying to generate cell lines carrying phosphomimetic substitutions. I have 5 primer pairs, each are ~60 bases.
I am using 2.5 micro Molar (uM) forward and the same for rev. primer amd 50ng template DNA. Also I have added 3% DMSO .
The PCR cycle I am following is
Initial denaturation at 98C for 1min
98C for 25 sec
72-70-68C for 25s
72C for 5m
final extension at 72 for 10m.
DpnI digestion for 2hrs at 37C.
I used competent E. coli. I have seen colonies (But also I have seen background in -ve ctrl plate!)
After mini-prepping and seq, I found I don't have any correct insertion. All of them were exactly like WT sequence!!
What do you think I should modify in this protocol to get correct insertions?
I would appreciate your add!
I am so sorry for not following your respectful comments.
I got all the mutants now!!
I have done two things which are the causes of previous failed mutagenesis.
1- I have used very very high annealing temperature, for PCR-based mutagenesis using long primers (~60 bp), I should use maximum 55C.
2- Pfu polymerase works perfectly, unlike Phusion polymerase, with blunt-end primers.
Thank you everybody for your suggestions!Following
- Jiexi Hou added an answer:5Does anyone know what will happen if there are two promoters in one construct?
pMDC85 promoter contains 2x35s constitutive promoter and I've put in another tissue-specific promoter down-stream of 35s. Now I am thinking what tissues in plants will GFP label?
Thanks Yuan-Yeu. I will let you know if I can get any good ideas after I read this paper.Following
- Leong Chia Choong added an answer:14Can anybody help with my ligation as it has failed multiple times(No colonies) ?
My goi is base on PCR cloning, as my primer design has 2 restriction enzyme site, which amplify my product with the sites (EcoRI and SapI). After digesting both my PCR product and plasmid, I ligated both of it with 3:1 vector to insert formula, and even tried with 6:1 but it is no working. My gene of interest is Kanamycin and my vector has ampicilin, so when doing selection on agar plate, the agar contain both the antibiotics.
Thank you for answering my question!
After multiple trial and error I have successfully ligated my product, and my previous failure might be due to that I have skipped a single step which is heat deactivation of my enzyme before transforming into competent E.coli cell.Following
- Charitha Gangadharan added an answer:7Can we use a lentiviral (promoter/ expression) construct for transfection in MCF7 cells by lipofectamine based method?
Can we use a lentiviral (promoter/ expression) construct for transfection in MCF7 cells by lipofectamine based method? If not how can I modify the construct for an antibiotic based selection construct? I plan to do a transient transfection with a GFP/LUC construct. The gene of my interest is already cloned in lentiviral construct. How can I subclone to a PEGFP/ promoter LUC construct?
Thank you all for the valuble inputs given and the time you took to answer my questionFollowing
- Sharmila Narayanan added an answer:7Can you help me with transient transfection?
We have cloned our gene in mammalian expression vector pEGFN1 vector by introducing a stop codon between the gene of interest and EGFP sequence so that no GFP is expressed. While performing transient transfection in HeLa and A-549, we used pEGFPN1 vector as control (Where GFP is expressed). It has been observed that the vector is also showing toxicity but slightly lesser than the gene of interest containing pEGFPN1. This we got to know by doing MTT after 48 hours of incubation. Does anyone have any idea, how can I solve this problem? Or what is going wrong that can be corrected.
@Paritosh . We isolate plasmid using kit (Sigma) and for transfection we use Serum free media onlyFollowing
- Andrew Jenkins added an answer:9Are you familiar with restriction digest with a single enzyme?
I am cloning out a fragment, using SacII, containing the ORF of my interest and cloning it into a SacII site in the multiple cloning site of my vector. The insert can obviously orientate itself in one of to ways in this case but I need it to be in the "correct" orientation. There are no other unique enzyme pairs flanking the ORF or in the MCS. Is there a way that I can encourage ligation of the "correct" product? Thank you.
If you're only getting the 'wrong' orientation - presumably meaning that the promoter transcribes the antisense strand - then it suggests that your product is toxic when overexpressed. You will need to keep the gene 'turned off' until it is time to induce it.
If for example, you are cloning in a plasmid of the pUC family with the gene under control of the lac operon, keep the lac operon turned off by adding glucose and leaving out IPTG. The drawback with this is that you lose the blue-white screening, but if you're using dephosphorylated vector this shouldn't be too much of a problem.
Another trick you might like to try is to deliberately pick small colonies. This will work if your product is mildly toxic and reduces growth rate without actually killing the cells.Following
- Nicolas Grandchamp added an answer:11Apart from HEK 293T cells, can HEK 293 also produce lentiviral vectors?
I use CaPO4 precipitation transfect 293T to produce lentiviral vectors(packing vectors are pMDL, pRev and pVSVG). However, I take the 293 other than 293T. So I am worry about whether 293 can produce the virus I need. Any suggestion will appreciated!
Agree with Aron, you can produce efficient LV with 293 but it is with 293T you will produce the most efficient LV.Following
- Mark K Chee added an answer:14What are the strategies for creation of constitutively active mutants of a protein?I am working on a protein which becomes active when phosphorylated at a particular tyrosine site. I am looking for methods/ strategies as how to make constitutively active mutant for this protein. Please suggest some.
An unnatural amino acid that mimics phosphotyrosine
- Rajendra Kumar Chauhan added an answer:7How can I check the large DNA sequence (in an expression vector) ?
I want to check gene sequence that is a large size (3.7kb). This gene is located in MCS of pQE9 vector (Qiagen).
How many primers that i should design for check full gene ?
Do i need to sequence double strand (using both forward primer and reverse primer)
Thank you for your suggestion
There are different programmes for sequencing a little bit longer stretches of sequences,We generally use Rapid for sequencing upto 700bp.The sequencing run takes a little longer than usual,but you get longer reads.So as other people have pointed to use 4-5 primer sets would generally solve your sequencing dillema.
Combination of primers from the vector and within your insert would be the best.Following
- Ravinder Kaundal added an answer:7Could anyone recommend a stable high transfection efficiency mouse cell line that I could use?I've been trying to transfect MEF cells using several methods and I did not get a very high efficiency (we have the Nucleofection kit too but there are many constructs that I need to transfect and it would be too expensive ) therefore I would like to try a different mouse cell line that shows a higher transfection efficiency. Could anyone suggest any?
I would truly appreciate your help. Thank you.
What was the transaction efficiency of MEF cell line with lipfectamine 2000?Following
- Bhanu P Jagilinki added an answer:15Is there any bacterial strain that can maximize the expression of recombinant protein?
We are trying to express a fusion of His tag protein in E. coli BL21 DE3 and Rosetta DE3 strains. Our fusion protein (~23 KDa in pET28a) does not appear.
The gene sequence is in frame with the His tag and we try change temperature and different concentrations of IPTG to induce the protein. Somebody have any suggestion for this problem?
Did u checked, if ur protein is going into inclusion bodies?? After sonication, centrifuge @15k rpm and load both pellet and supernatant separately.
For pET-28 constructs, prefer Rosetta-2DE3 where u have to add both Kanamycin and Chloramphenicol.Following
- Paola Campomenosi added an answer:16Is my gene amplifying?
I have a gene(wild type) cloned in pBAD18 Kanamycin resistant plasmid. The gene is 2185 bp. And the plasmid is 5437 bp. I wanted to insert a 6X His tag at the C-terminal end of the protein of this gene. I found that the last codon codes itself for a His. So I followed the general method for designing a primer i.e.: Last codon(CAT)-6X His codon (CAT or CAC)- Stop codon-XbaI recognition sequence. The primer length is 30 bp with a Tm of 62 degrees approx and the forward primer I used was 28 bp and has a EcoRI recognition site with Tm 59 degrees approx.
I have tried amplifying my gene using a gradient PCR vaying the annealing temperature (see gel image). I expect to get the product in between 2Kb and 3Kb(2203 bp). But I have a range of variation in amplification above 10 Kb. What is getting amplified is my question? Is my gene at all getting amplified? I dont think so. What is going wrong.
I am using Pfu polymerase with 30 steps amplification. I have done gradient PCR with variation in annealing please see gel image attached. PCR program:
Initial denaturation: 95 degree- 3 min
denaturation: 95 degree- 30 sec
annealing: gradient- 55- 64 degree- 30 sec
extension: 72 degree- 3 min 30 sec
Final extension: 8 min
Store: 4 degree.
Notice the amplified product that I am getting is above 10 Kb.- DNA Ladder used is 1Kb NEB ladder.
please suggest ways I can get my gene amplified for cloning. I want my insert to have a 5'-EcoRI and 3'-6X His-XbaI.
1) how much of your starting plasmid template do you use for PCR? Have you tried to load it on the agarose gel to check that the bands you observe are not the original template?
2) it seems to me that 30 nt are too short for your primer: it must contain 6 nt for the restriction site, 3 for the stop codon, 5(or 6) x3 His codons = 15-18 and finally the sequence complementary to your target... try to extend the complementary part at least to 18-20 nt.
(and beware you must not indicate a primer in bp, as it is single stranded! ;-))
- Daniel M Cohen added an answer:9Does pIRES2 express more EGFP than the mRNA before the IRES?
I've subcloned all of my mRNAs into pIRES2 (CMV:mRNA-IRES-EGFP) for expression in cell culture (CHO and TSA cells) and electrophysiology. I have been getting a lot of bright green cells with absolutely no measurable expression of my mRNA: about 50% of the green cells show the expected current, the other 50% react like an untransfected cell.
Is this a common problem with the pIRES?
The IRES-GFP cassette is designed to work in conjunction with a stop codon in your GOI. If this is absent, your GOI may not express properly. Based on what you describe, it sounds like the co-transfection experiment was a better setup. Perhaps the transfection efficiency of the IRES2-GFP plasmid is simply less good in your hands either due to increased plasmid size or due to variability in the quality of the purified DNA.Following
- Indrajit Sahu added an answer:3Can I convert the validated efficient shRNA sequence of one specific gene to siRNA sequence? If yes, how can I achieve it?
I have the validated efficient shRNA sequence targeted for one specific gene and its length is 21nt ,which is suitable for lentivirus knockdown pLKO.1 vector.
But now I want to knockdown this gene by siRNA or stable shRNA transfection by electroporation with pSUPER vector, for which I need the efficient 19nt sequence.
Can I convert the validated efficient shRNA sequence of one specific gene to siRNA sequence? If yes, how to achieve it?
YES, of course.
As per my knowledge is concerned if your 21-shRNA has efficiently worked, then you can directly convert into a 21 long siRNA. There is no need of modification at all. After all your shDNA-pLKO construct eventually converted into a single standard 21 base long siRNA, which had given u good results. And of course u just need to change the T to U.
Importantly, u need to keep the "2 base" (most preferably AT/AU) 3' overhang after the 19base antisense sequence. That will increase the activity (final degradation of mRNA by RISK-complex).
I have tried it in my lab and it works better.Following
- Touraj Farazmandfar added an answer:4What are the precise sequences for Age1 site to add to oligos for shRNA cloning in pLKO.1?
I'm planning to do shRNA cloning in the pLKO.1 for lentivirus production. i read the protocol provided by addgene et some others. There is a little that I don't understand regarding the Age1 restriction site. The protocol is based on annealed oligos that you put the open vector. The bases recommended to add at the 5' side of the forward oligo are CCGG. but the restriction site normally is CCGGT. Why we don't put the "T" at the end of this site whereas they put a full site for EcoR1? Hope i was clear . thanks in advance
order synthetic oligo cloned in your plasmidFollowing
- Nirav Rajivkumar Shah added an answer:1Is pSuper.neo.gfp vector a suitable system for stable microRNA over-expression or knockdown for its functional characterization?
I am planning to use pSuper vector system for over-expressing and knocking down a microRNA in breast cancer cells. Although it was initially designed for the purpose of siRNA expression, I am not quite sure about its suitability as far as miRNA expression is concerned. Also, I also would like to know that whether cloning a sequence antisense to a particular miRNA in the same system can efficiently result in knockdown of that miRNA.
I work with miRNA and to over-express, a particular miRNA first I amplified ~350 bp region containing pre-miRNA with my desired restriction site on both ends. I cloned this into pGEMT-Easy vector and then I cloned that piece into the PCDNA 3.1 + vector (zeocin). I transfected HCC-1954 cells with this cloned vector (0.5 ugs) and collected around 25 individual clones. It is extremely important to collect more clones since expression of your miRNA will vary from clone to clone. Based on Real time data, I have approximately 800-4000 fold up regulation of my miRNA of interest. For knock down the most suitable approach will be to use sponge. see this paper http://www.jbc.org/content/early/2013/08/06/jbc.M113.491803.full.pdf.
I hope this helps,
- Elena Azhayeva added an answer:1Has anyone used Trimer Codon Oligos and can offer advice?We want to create a semi-random expression library (around 30aa). This could of course be done by using poly-N oligonucleotides, but there is a newer technology where the oligo is synthesized using trimeric nucleotides (codons). This has advantages since you then can choose not to introduce stop-codons, avoid certain amino acids and make customized mixes of codons for certain positions in the oligo.
My question is has anyone experience with these? How long oligo did you order? Any company to recommend?
Metkinen Chemistry provide synthesis of the Trimer phosphoramidites as well as Randomized Oligonucleotide Libraries using them. The length of the oligonucleotide up to 100 b, but number of randomizations is limited to 10-14. You can get more information at www.metkinenchemistry.comFollowing
- Chiara Rapisarda added an answer:4Does anybody have a protocol for creating Stellar competent cells used in the In-fusion cloning kit?
I have already tested the reaction mix and it is useless. I think the trick is to use Stellar competent cells. Has anybody managed to produce a competent cell that was equivalent to the Stellar competent cells?
the in-fusion per se is not a problem (with or without enzymes it makes no difference to the final product). We make Top10 cells ourselves and I noticed that with the stellar competent cells I get a much better efficiency than with the top10 we make. We tried to use the stellar competent cells strain to make them competent again using our method but it didn't work. What strain do you use in your protocol? I will try it an compare it with the one we use.
- Sven Reister added an answer:10What is a suitable strategy for cloning cDNA open read frames > 2kb?
I want to clone the open read frame of some genes that are about 2.5kb in length so that I can then put them into some plasmids for overexpression. I have been trying and failing so far to get the ORF's amplified via PCR from isolated RNA from a range of animal tissues and also from cultured cells. I have tested the quality of the RNA by running it on an agarose gel and I get sharp clear ribosomal bands so the quality of my RNA im pretty happy with. I have also been able to amplify PCR fragments of the genes up to about 1600 using primers targeting different regions of the orf and even into the untranslated regions of the genes. When I try to amplify longer PCR products its usually unsuccessful. Therefore I am interested to hear from people who have successfully cloned genes of over 2kb from animal tissues or from cultured cells with regards what methods and kits etc have people used. I have been using tripure from roche for RNA isolation, the roche first strand cDNA synthesis kit and the roche fast start PCR kit.
If your clone is available @ addgene you drop to 60$ so have a look
- Monica Sharma added an answer:5Can anyone advise me on cloning and standardizing pQE30 in bacteria other than e coli M15?
any suggestions relating to the streak seen on the gel (run for plasmid) for the recombinants observed against the band for control plasmid.. and any detail in reference to the cloning process per se..can any one relate to
thanx all for your response ..will get back shortly.Following
- Gianmichele Massimo added an answer:43Can anyone recommend a good source for purchasing plasmids?I would like to buy plasmids from mouse.
My aim is to clone a protein of interest inserting a promoter and a tag (we already choose them and have them in the lab). We would then like to transfect cells (HEK cells).
I saw for example labome, but they are really expensive (400$).
Does anyone have any suggestions?
I have already checked and there isn't pCMV6 AC GFP plasmid....Following
- Angel L. Alvarez added an answer:2Where can I find the maximal DNA insert size of a vector?
Where can I find the maximal DNA insert size of vector?
And where can I find the amount of copy numbers of vectors?
The vectors I'm researching are: pDrive, pTrcHis TOPO TA, pcDNA3, pFastBac, pYAC3 and pET DEST 42.
Thats in advance!!
In addition, the copy number of a bacterial plasmid is determined by a key regulatory element which is present within the sequence, namely the "origin of replication". Distinct natural or synthetic origins of replication promote different copy numbers depending on the type and complexity of the regulation they exert. You can follow this link to find more info concerning the copy number associated to the most used ORIs. http://blog.addgene.org/plasmid-101-origin-of-replication. Luck !Following
- Kay-Dietrich Wagner added an answer:9How can I amplifying a 15kb fragment from a plasmid by PCR?
I try to amplify a 15kb fragment from a plasmid but can't manage to have any product yet. I tried to use Herculase II and condition suggested for product > 10kb but with no success. We don't have any kit to amplify such long DNA so I was wondering if some of you have a "home made" mix recipe of polymerases to help me to achieve this amplification as fast as possible?
Thanks for your help
As mentioned above Quick change II XL is good for doing this, I also used recently Herculase II, worked well. What Michael Rosemann mentioned might all help. I had sometimes bad experience with primer quality, also GC content is always an issue. If your primers have nearly the same Tm, what I normally do is to run 12 reactions on a gradient cycler without DMSO and 12 with DMSO, extension minimum 30s per kb. If this is not giving the correct band, then I change the primers (either same sequence purified or some base pairs extra to get a better GC content and more similar Tm). If you are in a rush it is faster to page purify the oligos yourself than to order HPLC quality from most companies.Following
- Kathryn M Edenborough added an answer:1Can you give me hints for the cloning of N1 NA into phw2000?
We are currently trying to clone some N1 NAs into the phw2000 vector using the method by Stech et al 2008. However, we seem to be getting either a high number of mutations or deletions of segments. Can anyone provide some hints of how you have cloned your NA into phw2000 in the past?
I modify pHW2000 by adding 3' and 5' 12nt of NA to the polI promoter or murine terminator respectively via PCR. In a separate reaction I amplify NA with universal primers and then use gibson cloning to insert the NA. This works well for me and avoids the use of restriction site cloning. Let me know if you need more details and I can give you primer sequences etc.Following
- Xiang Cheng added an answer:2Where should I order oligos for constructing tet on pLKO shRNA plasmids?
Hi all, where should I order 57 or 58bp oligo DNA for tet on plko shRNA construct? I try to order 57bp oligo for tet on pLKO shRNA from IDT and I get this information "Warning: Due to the length of this sequence, this oligo will be processed with a Complexity Service. A reduced yield and purity guarantee will apply.
Warning: Due to the length and complexity of this oligo, a Level II set-up fee and reduced yield and purity guarantees will apply. This oligo will also require additional review from IDT Customer Care. If the result of this review causes any change in pricing from what is displayed in your cart, a representative will notify you prior to processing.".
Does anyone get similar response from IDT? This seems that the 57bp oligo for shRNA contains specific structure and needs to be synthesized specially??? Do I need to
And the price for this 57 bp oligo from IDT is $139.82 and very high. can anyone give some suggestions? Thanks.
Yesterday I contacted the IDT company and they told me that there was a system error and the price should be around $15 for the 57bp oligo.Following
- Christopher R Heier added an answer:5Is it possible to do a genetic rescue if the shRNA was used to knockdown the coding sequence of the gene?
Hi All, I made a stable knock down cancer cell line for my gene and I want to do a genetic rescue by overexpressing it. But, my shRNA targeted the coding sequence of the gene and I found out it's better to target the 3'UTR region for follow up rescue experiment. Is it possible that my rescue might still work?
Or, if I increase/use a very high amount of DNA during transfection, will that help?
Or, can I do a transient knockdown followed by overexpression of my gene? Will it be considered a rescue?
Does anyone have any other suggestions on how can I make my experiment work without starting all over with a new shRNA?
- Luis Quintino added an answer:8How can I do the titration of the lentivirus with qPCR?
I have produced lentivirus with my gene of interest. the protocol refers to some papers(Kutner, R., Zhang, X.-Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nature Protocols 4, 495–505 (2009).) My question is how to perform the qPCR and analyze the result?(I can not understand the formula of this paper)
I purchased qPCR assay of GAG and RNaseP
Review of the previous experiments:
Day0: Seed 5*104 U87 cells/well in each 6 well plate.
Day1: Harvest 10ml supernatant containing LV from 293T cell culture. And concentrated to 100ul, split to 20ul/tube and store in-80’C. Dilute this stock 1ul in 99ul PBS, and add 0.5ul, 5ul, 50ul to designed U87 cell in 6well plate (each well has 1.2*105cell). One well is no infection ctr.(only add Polybrene).
Day2: Replace U87 cell medium to remove LV.
Day5: Harvest U87(96 after infection), and extract gDNA, then store in -20’C.
*qPCR assay of GAG and RNaseP(purchased from Life)
Result of qPCR
Mean Cq(GAG) Mean Cq(RNaseP)
0.5ul 29.71 23.95
5ul 26.95 24.01
50ul 23.97 24.11
* this is the Data analysis of the protocol:
"vector copy numbers in HOS cells are normalized to human RNaseP gene copies and presented as proviral copies per genome equivalent. Calculate titers (integration units per ml, IU/ml) according to the following formula:
IU/ml= C x N x D x 1,000)/V, where C = proviral copies per genome, N = number of cells at time of transduction (corresponding to about 1 x 105 HOS cells per well), D = dilution of vector preparation, V = volume of diluted vector
added in each well for transduction. "
summarize of my question:
1. What is the meaning of the 1st sentence? is that mean C value=2^(CqRNaseP-CqGAG) ???
2. This protocol dosen't need standard curve(I think). is the method which use standard curve is more accurate? now I have my plasmid of lentivector which contain GAG, so do I need to purchase a plasmid containing RNaseP?
3. Briefly speaking, how to analyze the qPCR result for LV titration?
Looking forward your help! thanks!
as the specific conditions of each cPCR reaction vary, we need to set one reaction as a normaliser. in our case the highest concentration of REF LV. In that reaction the dCT was 2.01. We then use this value to calculate the ddCT across the qPCR run.
Ref Titer is the tiger of the reference batch
Hope it helps,
- Nicolas Grandchamp added an answer:6Has anyone got a protocol for lentiviral transduction of B-CLL cells?
I have been trying to transduce primary CLL cells with virus made using psPAX2 and pMD2.G plasmids. The virus I have generated works because I have managed to transduce HEK cells as well as the CLL cell line MEC-1. However, I haven't had any success with primary CLL cells. I have tested different polybrene concentrations, concentrating the viral supernatant in an ultracentrifuge as well spinnoculation without any luck.
If anyone has any suggestions or even better a protocol that they would be willing to share I would be most grateful.
As described through the link I put, I advise you to transduce cell in 24-well plate in a volume of 200ul with 10^2-10^4 cells.
About the promoter CMV is not a realy good promoter in your experimental context but Emu should be.
I advise you also to do a condition with DEAE-D
Let me know if you have other question or about the results of your next transductions!
About Molecular Cloning
A platform to ask questions and discuss the procedures involved in Molecular Cloning.