Guf

Hello,

I have a problem. I need to determinate concentration of MS2 phage-like particles (they do not form plaques). I found that some authors use correlation between absorbance, molecular weight and "extinction coefficient" to determine number of MS2 phage-like particles in the solution:

citation: The number of AR2 particles may be determined using the extinction coefficient of 1 OD₂₆₀=
8 mg/ml of MS2 bacteriophage and the molecular weight is 3 x 10⁶. Based on this procedure, approximately 1 x 10¹⁶ MS2 phage-like particles can be purrified from one liter of cultuire.

another citation: The concentration of MS2 phage-like particles was determined using an extinction coefficient of 8 mg/ml of MS2 bacteriophage per absorbance unit at 260 nm and a molecular weight of 3.0 x 10⁶.

Please, what is the exact method of calculation, I'm not a mathematician. I need to know how many MS2 phage-like particles (10³ or 10⁶) are present in the solution. Could you give me an example of calculation, please? Thank you.

Hello,

I need to amplify 7 kb sequence with high fidelity. We have three high-fidelity polymerases in our lab:


PfuUltra High-Fidelity DNA polymerase (from QuikChange II site directed mutagenesis kit, Agilent)

KAPA Hifi HotStart DNA polymerase (Kapabiosystems)

Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs)


Naturally, each manufacturer claims that his product is the best one. I have no experiences with HF polymerases so would it be possible to advise me which polymerase is really the best, please? Thank you.

Hello, I need your recommendation. I need to digest my plasmid and PCR product with 2 different endonucleases, BlpI (10 000 U/ml) and HindIII (100 000 U/ml). Both of them are from NEB.

According to Double Digest Finder (https://www.neb.com/...e-digest-finder) I should perform digestion in NEBuffer 2 at 37 °C.

And my question is how to set up the reaction?

I found that restriction mixture composition:

DNA - up to 1 ug
Buffer (10x) - 2 ul
Enzyme 1 (10 U/ul) - 1 ul
Enzyme 2 (10 U/ul) - 1 ul
H2O - up to 20 ul

What do you thing? Second question is: How long to digest it? Are 2 hours sufficient for digestion?

Is it possible to dilute HindIII (100 000 U/ml) to working concentration (10 000 U/ml) in water? Or I should to choose something else, for example NEBuffer?

BlpI can not be heat inactivated so after digestion I have to purify the mixture. Is it better to purify it with PCR purification kit or use Gel extraction kit? Thank you :-)

Hello everybody,

I´m new here and need you advice. This is my problem:

I want to clone specific sequence into the specific vector position. The position is the HindIII restriction site. I created my own specific sequence and let it synthetized, This sequence has 170 bp in lenght. After that I designed primers, which introduce HindIII restriction sites to the ends of 170 bp sequence.

Here is the beginning of forward primer: 5´-GAGAAAGCTTC.... -3´ (HindIII restriction site is in bolt).

Here is the beginning of reverse primer: 5´- TAGGAAGCTTG... -3´ (HindIII restriction site is in bolt).

After optimization of PCR I obtained amplicons which should contain HindIII restriction sites at the ends. Purification of PCR product was performed with QIAquick PCR Purification Kit according to manufacturer guideline. Than I measured concentration and purity of PCR product on nanodrop: c = 120 ng/microliter and 260/280 nm = 1,83, 260/230 nm = 2,6.

Than i performed overnight restriction digestion with HindIII: 40 microliters of purified PCR product (cca 4800 ng DNA), 31 microliters of water, 8 microliters of buffer B and 1 microliter of HindIII (100,000 U/ml). Purification of digested PCR product was performed next day using again QIAquick PCR Purification Kit, measure on nanodrop gave the following values: c=127 ng/microliter and 260/280 nm = 1,81, 260/230 nm = 1,86. Digestion of PCR product with HindIII  was verified by agarose gel electrophoresis, digested PCR product was smaller then undigested.

Than I isolated vector from bacterial culture using Macherey-Nagel NucleoSpin Plasmid Kit according to manufacturer instructions.  Than I measured concentration of vector on nanodrop:  c = 92 ng/microliter and 260/280 nm = 1,86 and 260/230 nm = 1,99. Than i performed overnight restrition digestion with HindIII: 11 microliters of vector (cca 1000 ng DNA), 6 microliters of water, 2 microliters of buffer B and 1 microliter of HindIII (20,000 U/ml).

Next day I performed dephosphorylation. I added CIAP directly to the restriction mixture. I added 0,5 microliter of CIAP and let the tube stay in room tempetature for 30 minutes and after I added next  0,5 microliter of CIAP and let it stay for another 30 minutes. (I use this CIAP from NEB: http://www.neb.com/n...roductm0290.asp ). It is good approach how to dephosphorylate the vector or not?

After dephosporylation i loaded the mixture into the agarose gel (GTG SeaKem) and extracted the digested and dephosphorylated vector with QIAquick Gel Extraction Kit according to manufacturer instructions. Than I measured concentration and purity of vector on nanodrop: c = 9,5 ng/microliter and 260/280 nm = 2,3, 260/230 nm = 0,02. But a nice band was clearly visible on control agarose gel...


According to NEB Quick Ligation Kit (http://www.neb.com/n...roductM2200.asp) I prepared ligation mixtures and after that I tried to transform E. coli BL21 (DE3). Than I selected potential tranfromants with kanamycin LB plates (30 micrograms/microliter). But i did not acquire colonies.

So I tried to do another lagation mixtures, but still nothing grows. So i tried this mixture (ratio 1:5, vecrot:insert), insert has 154 bp and vector 7100 bp:

5,2 microliters of vector ( 50 ng DNA)
3 microliters of insert (PCR product, 3,3 ng DNA)
1,86 microliters of water
10 microliters 2x ligation buffer
1 microliter of ligase

and let it ligate in 16 °C overnight

Than I tried to transform E. coli BL21 (DE3) and selected potential tranfromants with kanamycin LB plates (30 micrograms/microliter). After 2 day of cultivation I obtained some big collonies and a lot of small colonies. The small colonies are not resistant against kanamycin when they are tranferred into liquid LB broth with kanamycin. The big colonies are resistant but they do not contain recombinant plasmid :-(

Would it be possible to tell me what is wrong? Thanks. :-)