Paul.xu at 14:54, 10 August 2019

2019-08-10T14:54:11Z

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== Bovine Thrombin ==

Sigma T4648

Thrombin from bovine plasma lyophilized powder, 40-300 NIH units/mg protein (biuret)

4648-1KU $55.60

84 ug/ml--> 3.7 ug/ml to 150 ug/ml, such as 25 ug/ml in 50 ul reaction

== BSA ==

25 ug/ml or 50 ug/ml

= reverse-transcriptase reaction =

use 0.5 ul of Ectoine* (0.71 g/mL) in 5 ul total vol 

== PT for qPCR ==

1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer)

https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-11-41

== TCN ==

optimal: 0.6 mol/L d-(+)-trehalose, 0.48 mol/L l-carnitine, and 0.8% NP-40

for blood, add heparin

[https://jmd.amjpathol.org/article/S1525-1578(10)60043-8/fulltext https://jmd.amjpathol.org/article/S1525-1578(10)60043-8/fulltext]

l-Carnitine inner salt (C0158), d-(+)-trehalose dehydrate (T9531), betaine (B2629), heparin (H3393), and Nonidet P-40 (NP-40) were purchased from Sigma-Aldrich (St. Louis, MO). Three commercial PCR enhancers, GC Solution (used with FastStart Taq), Q-Solution (used with HotStarTaq Plus), and Hi-Spec Additive were obtained from Roche Applied Science (Indianapolis, IN), QIAGEN (Germantown, MD), and Bioline (Taunton, MA),

[https://www.tcichemicals.com/eshop/en/us/commodity/C2625/cmp/cts/ https://www.tcichemicals.com/eshop/en/us/commodity/C2625/cmp/cts/]

OmniTaq (OT) Omni Klentaq (OKT)

Conventional PCR

PCR using OmniTaq or Omni Klentaq was performed in a 50-μl volume in 0.5-ml or 0.2-ml tubes. The reaction buffer contained 50 mmol/L Tris-HCl, pH 8.3 (OT) or 9.2 (OKT), 16 mmol/L ammonium sulfate, 0.1% (v/v) Tween 20, and 2.5 mmol/L (OT) or 3.5 mmol/L (OKT) magnesium chloride. The reactions contained 2 U of OT or OKT enzyme, 0.2 μmol of each primer, and 200 μmol of dNTP mix.

d-(+)-trehalose : 0.5 M
l-carnitine 0.12 to 1.2 M,

Sample: 10% (final concentration of total PCR volume) whole blood, serum, heparinized plasma, and plasma treated with citrate or EDTA, using an OT/OKT mix (1:1)

d-(+)-trehalose alone increased the PCR yield, but it did not help much in improving specificity, especially with a GC-rich target

Trehalose stimulated amplification of relatively easy DNA targets, but for more difficult and GC-rich targets the addition of l-carnitine was crucial. The detergent NP-40 may help lyse the blood cells or viral capsids in crude clinical samples. None of the PEC components alone was sufficient to elicit the potent enhancing effect observed with the triple combination, especially with crude samples and GC-rich targets.

the enhancing effect of betaine was target-dependent. PEC was able to lower the melting temperature as well and outperformed betaine with GC-rich targets
[[Category:Protocols]]

PCR Enhancer - Revision history

Paul.xu at 14:54, 10 August 2019