2038 pL42-sso7d-KlenTaq

Cloning of 2038 pL42-sso7d-KlenTaq

Purpose: Cloning of2038 pL42-sso7d-KlenTaq==

Date: 2014-2----

Notebook:0

Page:

Clone ID:2038

Experimenters:  shanyi

Results

CLONING

subclone from 377 pET30b-sso7d-Taq_d289 using Nde1 + EcoR1 to 1984 pL42

Colony Screening

Date: 2014_2_14

Make miniprep, digestion with Nde1 + EcoR1

Purification

use 5 ml Ni column, linear gradient. mw =70

Purification method 2

Cook at 75 oC for 30 min, Spin down, load to Ni column, elute with linear gradient from 10 to 300 mM I. Combine elution peak fractions. Cocentrate using spin column. dialysis vs 20 mM Tris-HCl pH 8.0, 50 mM NaCl buffer. Load to 1.6 X10 cm Q HP column preequlibrated with 50 mM NaCl, 20 mM Tris-HCl, pH 8.0, elute with linear gradient from 50 mM to 1 M NaCl.

Protein eluted at ~ mM NaCl.

1	2	3	4	5	6	7	8	9	10	11	12
				Taq	Taq	Taq	Taq

Purity of Taq (Lane 5, 6, 7, 8) > 95%

Combine fractions containing Taq protein, Concentrate and dialyse vs Dialyssis buffer containing 50% glycerol,---

Final Protein concentration is 1.5 mg/ml, total 6 ml (9 mg)

Make serial dilution for PCR, use 1 ul diluted protein for 25 ul PCR.

Lane 1	2	3	4	5	6	7	8
1:5 dilution	1:7.5	1:10	1:12.5	1:15	1:20	1:30	Kfu

Usually Taq has 80,000 units/mg protein.

Looks like 1:5 dilution works, assuming 1 ul diluted protein has 2.5 units, 6 ml 1.5 mg/ml stock --> 30 ml of 2.5 units/ul working Taq (for 25 ul reaction. For 50 ul reaction 1:2.5 dilution should be used

Total is only 75,000 units to 100,000 units from 8 L culture.

For customers: dilute 1:2.5 ?

Protein Purification

Protocol

Result

SDS-PAGE gel

looks like 1:100 dilution works. Assume 1ul diluted protein equals to 1.25U

specificity activity is around 78000U/mg.

Final concentration 5.82mg/mlX2.5ml.

Protein Purification

SDS-PAGE