Dear all, please give information what are disadvantage using dephosphorylation vector (5-OH). what kind literature and theory related about that
Hi there,
Vector dephosphorylation avoids vector self-ligation event during cloning but it imposes the use of phosphorylated insert (which is the case when insert is generated by ER digestion).
As Dominique said, the dephosphorylation of vector helps in preventing vector self ligation during cloning step. In that case you need to do kinase treatment to your insert to get phosphorylation as it is necessary of ligase enzyme to ligate your vector and insert. In case if you are digesting your insert after PCR then you may not need to do kinase treatment for insert because it already have phosphate ends.
how to prepare phosphorylated insert DNA from PCR product?? do you hve suggestion, I Confucius converse PCR product into pmol.
For insert phosporylation :
if the PCR product is digested by restriction enzymes, it is 5' phosphorylated. If using PCR product directly for ligation, you have to phosphorylate it using Polynucleotidekinase (PNK).
do you know the protocol phosphorylate pcr product?? what kind the step? I phosphorylate my primer PCR or I phosphorylate my PCR product?? do have suggestion?
please refer this link
https://www.neb.com/~/media/Catalog/All-Products/96F1AABD36E2412F858AB6375181E2C2/Datacards%20or%20Manuals/M0201Datasheet-Lot0911207.pdf
simple protocol is, generally if you digest your PCR product with restriction enzyme (note: if you add restriction enzyme sites in your primers that used for insert amplification) that generate phosphate end in this case you no need to do PNK treatment. but if you are going to do blunt end cloning (if you are not digest with restriction enzymes) then your insert need to get phosphorylaion by PNK treatment.
so follow this protocol if you have NEB PNK enzyme or say which brand enzymes you are having in lab.
Protocol 1: Non-Radioactive Phosphorylation with T4 PNK
Set-up the following reaction:
MQ- adjust to 50 µL
10 X T4 PNK buffer -5 µL
insert -10-30 µL (50-100 ng/ul)
10mM ATP-5 µl
T4 PNK enzyme-1 µL
Total value adjusted to -50 µL
Protocol 2: if you have T4 DNA ligase buffer then you can use it for PNK treatment reaction because it have ATP so no need to add separately.
Reagent
Volume
MQ- adjust to 50 µL
10 X T4 buffer -5 µL
insert -10-30 µL (50-100 ng/ul)
T4 PNK enzyme-1 µL
Total value adjusted to -50 µL
Incubate at 37°C for 30 minutes.
After PNK treatment then again you can do PCR column purification elute your insert in 10- 30 ul use for ligation reaction or ethanol precipitation, then dissolve precipitated insert in 20 ul of sterile water or 1 X TE buffer then use it for ligation.
1X T4 DNA Ligase Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylation (T4 Polynucleotide Kinase exhibits 100% activity in this buffer).
For more information see NEB website
How about inactivate kinase, I read in NEB website it is need Heat Inactivation
65°C for 20 min, when you inactivate ?
How about your insert? do you directly phosphorylate after PCR or you phosphorylate after purification PCR by gel extraction?
if your doing gel extraction after PNK treatment then no need of heat inactivation. but still you want to do means then you could do after PNK treatment. After that you do gel extraction/ column purification.
I have question about PCR product was phosphorylated, it was amplified using the phosphorylated primer or I dont need phosphorylated my primer?
If both primers are phosphorylated then your PCR product is phosphorylated.at both 5'ends
it is okey if I use unphosphorylated primer but my PCR product I continue with PNK treatment.., which more better, using primer phosphorylated or PCR product treat PNK after amplified ?
i think its better if you perform a PNK treatment of your PCR product.
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