การสกัดพลาสมิด (Plasmid Extraction)

α+ Solution™ High Yield Plasmid Extraction Mini Kit

Name: α+ Solution™ High Yield Plasmid Extraction Mini Kit
Brand: ALPHAGEN
SKU: APHPD004 / APHPD100 / APHPD300

รหัสสินค้า: APHPD004 / APHPD100 / APHPD300

ผู้ผลิต: ALPHAGEN

ขนาด: 4 preps100 preps300 preps

ราคา: กรุณาสอบถาม

สอบถามราคา / สั่งซื้อ คู่มือ (PDF)

หลักการ: mini spin column (silica matrix)
ปริมาณตัวอย่าง: 1 ~ 5 ml
เวลาที่ใช้: < 25 minutes

การสกัดพลาสมิด (Plasmid Extraction)

Ordering information

APHPD004	4 preps
APHPD100	100 preps
APHPD300	300 preps

Component

	APHPD 004	APHPD 100	APHPD 300
PD1 Buffer	1.5 ml	30 ml	90 ml
PD2 Buffer	1.5 ml	30 ml	90 ml
PD3 Buffer	1.5 ml	40 ml	120 ml
PDW Buffer (concentrate)	1.3 ml	35 ml	98 ml
Wash Buffer (concentrate)	1.0 ml	20 ml	50 ml
Elution Buffer	0.5 ml	15 ml	35 ml
PD Column	4 pcs	100 pcs	300 pcs
Collection Tube	4 pcs	100 pcs	300 pcs
RNase A (Lyophilized)	0.15 mg	3 mg	9 mg
User Manual	1	1	1

Brief Prodecure

Specification

※ Principle : mini spin column (silica matrix)

※ Sample size : 1 ~ 5 ml

※ Size of plasmid or construct : < 15 kb

※ Operation time : < 25 minutes

※ Typical Yield : 25 ~ 40 µg

※ Binding capacity : 60 µg/ column

※ Column applicability : centrifugation and vaccum

Important Notes:

※ Store RNase A at -20 °C upon recipit of kit.

※ Add 0.5 ml of PD1 Buffer to a RNase A tube, Dissolve the RNase A by vortexing. Briefly spin the tube and transfer the total RNase A mixture back to the PD1 bottle, mix well by vortexing and store the PD1 buffer at 4 °C.

※ If precipitates have formed in PD2 Buffer, warm the buffer in 37°C waterbath to dissolve precipitates.

※ Preparation of PDW Buffer and Wash Buffer by adding 96 ~100% ethanol (not provided) for first use.

※ Centrifugation steps are done by a microcentrifuge capable of the speed at 11,000 ~1,8000 x g.

Trouble shooting

Problems	Possible reasons	Solutions
Low yield	Bacterial cells were not lysed completely	Too many bacterial cells were used (OD600 > 10). Separate the bacterial culture into multiple tubes. After PD3 Buffer addition, break up the precipitate by inverting to ensure higher yield.
	Overgrown of bacterial cells	Incubation time should not be longer than 16 hours.
	Bacterial cells were insufficient	Ensure that bacterial cells have grown to an expected amount (OD600 > 1) after incubation under suitable shaking modes.
	Incorrect DNA elution step	Ensure that Elution Buffer was added and absorbed to the center of the PD Column matrix.
	Incomplete DNA Elution	If the size of DNA fragments is larger than 10 kb, use a preheated Elution Buffer (60~70°C) on the solution step to improve the elution efficiency.
	Incorrect preparation of PDW Buffer and Wash Buffer	Ensure that the correct volume of ethanol (96 ~ 100 %) was added to PDW Buffer and Wash Buffer before use.
Eluted DNA does not perform well	Residual ethanol contamination	After Wash Step, dry the PD Column with an additional centrifugation at top speed (~18,000 x g) for 5 minutes or incubation at 60°C for 5 minutes.
Genomic DNA Contamination	Lysate prepared improperly	Gently invert the tube after adding the PD2 Buffer. And the incubation time should not be longer than 5 minutes. Do Not use overgrown bacterial culture.
RNA Contamination Plasmid DNA	Insufficiency of RNase A activity in PD1 Buffer because of long-term storage	Prior to using PD1 Buffer, ensure that RNase A was added. If RNase A added PD1 Buffer is out of date, add additional RNase A into PD1 Buffer to a concentration of 50 μg/ ml then store 4°C. Too many bacterial cells were used, reducing sample volume.
Smearing or degrading of Plasmid DNA	Nuclease contamination	If used host cells have high nuclease activity (e.g., enA⁺strains), perform the following optional Wash Step to remove residuary nuclease. a. After DNA Binding Step, add 400 μl of PDW Buffer into the PD Column and incubate for 2 minutes at room temperature. b. Centrifuge at full speed (~18,000 xg) for 30 seconds. c. Proceed to step 6.2.
Plasmid DNA is not adequate for enzymatic digestions	Eluted plasmid DNA contains residual ethanol	Make sure you have discarded the flow-through after washing with Wash Buffer (Step 6.2) and centrifuged for an additional 3 minutes (Step 7).
Denatured Plasmid DNA migrate faster than supercoiled form during electrophoresis	Incubation in PD2 Buffer too long	Do not incubate the sample longer than 5 minute in PD2 Buffer

Documents

Protocol (PDF)

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