Choosing Input Amounts for the Monarch HMW DNA Extraction Kits

Cells  


The sample input range is 1 x 105–1 x 107 cells, but an input amount of 1 x 106 cells is recommended. The upper limit for cell input amounts is dictated by the viscosity of the lysed sample, which poses a challenge to the efficiency of the enzymes, precipitation onto the beads, and the dissolving of the purified DNA. If using more than 2 x 106 cells, purified DNA samples will be viscous and may be more difficult to dissolve and handle. If using an input below 1 x 105 cells, DNA recovery will be significantly less efficient. It is important to note that if employing a low agitation speed during lysis, inputs should not exceed 5 x 106 cells. When working with samples less than 5 x 105, follow protocol guidance for Low Input to ensure the buffer volumes used reflect the lower cell count. 

Blood 


Mammalian Blood 

The sample input range for mammalian blood samples is 100 µl up to 2 ml. Working with a starting sample of 500 µl is recommended for most sample types; 200 µl is recommended for rabbit samples, which have a high cell content. If working with starting volumes > 500 µl, samples will need to be initially processed in larger tubes or split into 500 µl aliquots. The container needs to accommodate the addition of 3 volumes of RBC Lysis Buffer; working with 500 µl enables processing in a 2 ml tube. If splitting the sample into multiple aliquots, the pellets can be combined into a single 2 ml tube after RBC lysis to facilitate further processing. If working with sample volumes below the recommended input amount (500 µl for most samples), follow the protocol guidance for Low Input to ensure the buffer volumes reflect the lower cell count. 

Nucleated Blood 

The maximum input for successful DNA extraction from nucleated blood is 20 µl; exceeding this will overload the system. The recommended input amount is 5 µl.  

Table 1: Guidance on sample input amounts and expected results: cells & blood (NEB #T3050)

 
Table 1 provides data on minimum, maximum, and recommended input amounts for various cell lines and blood samples using the Monarch HMW DNA Extraction Kit for Cells & Blood. Data on yield, purity, RNA content and DIN values is also provided. Samples that were successfully tested in standard ligation-based Oxford Nanopore Technologies sequencing runs are indicated. RNA content was determined by HPLC analysis of nucleoside content after digestion of 1 µg of eluted nucleic acid with the Nucleoside Digestion Mix (NEB #M0649). Yields from blood samples vary by donor due to different leukocyte content; yield can vary up to 3-fold by donor. Similar yield and purity results were obtained with different anticoagulants (e.g., EDTA, citrate, heparin and PAXgene Blood DNA tubes were tested).  

Using input amounts below the recommended minimum will reduce yields drastically. Exceeding maximum input amounts will result in DNA eluates that are highly viscous and difficult to dissolve and will reduce purity of the isolated DNA. Results are shown for samples that were lysed with agitation at 2,000 rpm.  

MINIMUM INPUT (CELLS)

MAXIMUM INPUT (CELLS)*

RECOMMENDED INPUT AMOUNT (CELLS)

YIELD (μg) FROM 1 x 10CELLS

PURITY RATIOS

RNA CONTENT

VALIDATED FOR ONT SEQUENCING?

A260/280

A260/230

HEK293

1 x 105

1 x 107

1 x 106

11.5–13

1.86

2.4

≤ 1%

Yes

HeLa

1 x 105

1 x 107

1 x 106

12.9

1.86

2.4

≤ 1%

Yes

NIH3T3

1 x 105

1 x 107

1 x 106

9.4

1.86

2.4

≤ 1%

Yes

Jurkat

1 x 105

1 x 107

1 x 106

13.7

1.86

2.5

≤ 1%

Yes

K562 (suspension cells)

1 x 105

1 x 107

1 x 106

13.7

1.86

2.4

≤ 1%

Yes

HCT116

1 x 105

1 x 107

1 x 106

16.9

1.86

2.5

≤ 1%

Yes

A549

1 x 105

1 x 107

1 x 106

12.7

1.86

2.3

≤ 1%

Yes

U5Os

1 x 105

1 x 107

1 x 106

10.6

1.86

2.4

≤ 1%

Yes

HepG2

1 x 105

1 x 107

1 x 106

13.4

1.81

2.2

≤ 1%

Yes

NCI-460

1 x 105

1 x 107

1 x 106

9.5

1.86

2.4

≤ 1%

Yes

SK-N-SH

1 x 105

1 x 107

1 x 106

9.5

1.86

2.4

≤ 1%

Yes

Aa23

1 x 105

1 x 107

1 x 106

8.7

1.81

2.3

≤ 1%

Yes

Mammalian Blood

MINIMUM INPUT (μl)

MAXIMUM INPUT ((μl)*

RECOMMENDED INPUT AMOUNT (μl)

YIELD (μg) for 500 µl**

PURITY RATIOS

RNA CONTENT

VALIDATED FOR ONT SEQUENCING?

A260/280

A260/230

Human***

Fresh

100

2,000

500

12–32

1.86

2.4

≤ 1%

Yes

Frozen

100

2,000

500

9–30

1.86

2.4

≤ 1%

Yes

Mouse

Fresh

100

2,000

500

7–11

1.88

2.4

≤ 1%

Yes

Frozen

100

2,000

500

16–17

1.88

2.4

≤ 1%

ND

Rat (fresh only)

Fresh

100

2,000

500

29–38

1.87

2.4

≤ 1%

Yes

Rabbit

Fresh

100

500

200

12–15

1.72

1.9

≤ 1%

Yes

Fresh

100

500

200

200 µl: 4–5

1.89

2.4

≤ 1%

Yes

Frozen

100

500

200

200 µl: 4–5

1.89

2.4

≤ 1%

Yes

Pig

Fresh

100

2,000

500

up to 42

1.86

2.4

≤ 1%

Yes

Frozen

100

2,000

500

up to 40

1.86

2.4

≤ 1%

Yes

Horse

Fresh

100

2,000

500

16

1.86

2.3

≤ 1%

Yes

Frozen

100

2,000

500

22.3

1.86

2.4

ND

ND

Cow

Fresh

200

2,000

500

7

1.86

2.4

≤ 1%

Yes

Frozen

200

2,000

500

9.1

1.86

2.4

ND

ND

Rhesus monkey

Fresh

100

2,000

500

52

1.86

2.4

≤ 1%

Yes

Frozen

100

2,000

500

52.6

1.86

2.5

ND

ND

Goat

Fresh

100

2,000

500

24

1.87

2.4

≤ 1%

Yes

Nucleated Blood

MINIMUM INPUT (μl)

MAXIMUM INPUT (μl)*

RECOMMENDED INPUT AMOUNT (μl)

YIELD (μg) per 5µl

PURITY RATIOS

RNA CONTENT

VALIDATED FOR ONT SEQUENCING?

A260/280

A260/230

Chicken

Fresh

2

20

5

33

1.86

2.5

ND

Yes

Frozen

2

20

5

30

1.86

2.5

ND

ND

Turkey

Fresh

2

20

5

37

1.87

2.4

ND

Yes

Frozen

2

20

5

28

1.87

2.5

ND

ND

ND = Not determined

*    For low agitation speeds, do not exceed 5 x 106 cells
**  Unless otherwise stated
*** Compatible with K2-EDTA, Na-citrate, Na-heparin, PAXgene®  Blood DNA


Figure 1: Linear correlation between yield and input for cells and blood.  



Summarized yield data for HMW DNA preps are shown carried out at 2,000 rpm during lysis, using HEK293 cultured cells and fresh human blood samples from different donors as input material in the corresponding protocols. The starting materials were diluted to 5 different concentrations to cover the entire recommended input range. Cell samples ≤ 5 x 105 cells and blood samples <500 μl were purified using the recommended volumes for low input samples. Obtained yields show a high degree of linearity over the displayed input range.   

 

Tissue

 
The sample input range is 5–25 mg for most tissues (2–15 mg of DNA-rich / soft organ tissues (e.g., kidney, liver), 5–20 mg for brain). The upper limit for tissue input amounts is often limited by the viscosity of the lysed sample, which negatively impacts enzyme access, protein removal, precipitation onto the beads, and dissolving/resuspension of the purified DNA. In some samples, the high amounts of fibers or fatty acids can be factors that limit the input amounts. If a lower-than-recommended input amount is used, DNA recovery will be significantly less efficient. Standard and low input protocols are provided to ensure the buffer volumes are appropriate and that precipitation onto the beads is efficient. If working with fatty or fibrous tissues (e.g., brain and muscle), and only very small amounts of sample are available (< 5 mg), see guidance below. 

Using Very Low Input Amounts 

In some cases, inputs below the range provided can be successfully processed using this kit if the volume of lysate, reagents and buffers are reduced, as described below. For brain and muscle samples, input amounts of 2–5 mg have been successfully processed. When working with samples in this input range, stop agitation after 15 minutes during the 45-minute lysis for maximum yield. 

  • Part 1, Step 1: use 100 µl Tissue Lysis Buffer and 10 µl Proteinase K
  • Part 1, Step 6: use 5 µl RNase A 
  • Part 1, Step 8: use 55 µl Protein Separation Solution
  • Part 2, Step 2: use 90 µl isopropanol  

Bacteria 


The sample input range for E. coli is 5 x 108 – 5 x 109 cells. As described for tissue samples, the upper limit for bacteria input amounts is limited by the viscosity of the lysed sample, which negatively impacts enzyme access, protein removal, precipitation onto the beads, and dissolving/resuspension of the purified DNA. If an input amount below the recommended amount is used, DNA recovery will be significantly less efficient. Standard and low input protocols are provided to ensure the buffer volumes are appropriate for the sample input amount used. The sample input range for B. cereus using the low input protocol is 2 x 108 – 4 x 108 cells. 

Table 2: Guidance on sample input amounts and expected results: tissue, bacteria and other samples (NEB #T3060) 

The table below provides guidance on the minimum, maximum, and recommended input amounts for various sample types when using the Monarch HMW DNA Extraction Kit for Tissue. Data on yield, purity, RNA content and DIN values is also provided. Samples that were successfully tested in standard ligation-based Oxford Nanopore Technologies sequencing runs are indicated. Using input amounts that exceed the maximum will lead to challenges in solubility and viscosity, and purity may be affected. If more starting material is required, splitting the sample and performing multiple preps is recommended. RNA content was determined by HPLC analysis of nucleoside content after digestion of 1 µg of eluted nucleic acid with the Nucleoside Digestion Mix (NEB #M0649). Using input amounts below the recommended minimums will reduce yields drastically.  

MINIMUM INPUT (CELLS)

MAXIMUM INPUT (CELLS)*

RECOMMENDED INPUT AMOUNT (CELLS)

YIELD (µg) FOR RECOMMENDED INPUT (YIELD PER mg)

PURITY RATIOS

RNA CONTENT

VALIDATED FOR ONT SEQUENCING?

A260/280

A260/230

Mammalian Tissue

Mouse brain

Fresh

2**

20

15

12–21

1.87

2.39

ND

YES

Frozen

2**

20

15

15–21 (1–1.5)

1.86

2.48

ND

YES

Mouse liver

Fresh (w/NaCl)

2

15

10

7

1.84

2.10

1.2%

YES

Frozen (w/NaCl)

2

15

10

17–19 (1.7–1.9)

1.89

2.50

ND

YES

Fresh*

2

15

10

20

1.84

1.52+

8.7%

YES

Frozen*

2

15

10

27–31 (2.7–3.1)

1.89

1.93++

ND

YES

Mouse muscle

Fresh

2**

25

20

8–9

1.87

2.25

2.1%

YES

Frozen

2**

25

20

12–16 (0.6–0.8)

1.87

2.30

ND

YES

Mouse kidney

Fresh

2

15

10

23–34

1.86

2.44

ND

YES

Frozen

2

15

10

32–41 (3.2–4.1)

1.86

2.53

0.8%

YES

Mouse tail

Frozen

2**

25

20

20          (1.8–2.1)

1.86

2.43

ND

YES+++

Mouse ear punch

Fresh

2**

15

10

15–16 (1.5–1.6)

1.86

2.29

ND

YES

Rat kidney

Frozen

2

15

10

20–25

1.87

2.40

ND

YES

Bacteria

E. coli
(Gram-negative)

Frozen

5 x 108 cells

5 x 109 cells

1 x 109 cells

8–9

1.89

2.31

1.7%

YES

B. cereus
(Gram-positive)

Frozen

2 x 108 cells

4 x 108 cells

2 x 108 cells

4–5

1.86

2.20

3.9%

YES

M. luteus
(Gram-positive)

Frozen

ND

ND

1 x 108 cells

2.0

1.89

2.09

ND

ND

Amphibian

X. laevis

Fresh

ND

ND

3–4

5

1.86

2.51

2.3%

ND

Yeast

S. cerevisiae

Fresh

ND

ND

3.8 x 108 cells

15

1.90

2.01

ND

ND

Insect

A. aegypti

Frozen

ND

ND

15

6

1.84

2.53++

2.7%

ND

ND = Not determined

*     Standard protocol without recommended NaCl treatment
**    If working with input amounts <5 mg, refer to the product manual for guidance on reducing buffer volumes
+     Measured with Nanodrop One; systems that differentiate turbidity in the content profiling will give higher values
++   Measured with Unchained Labs Lunatic (formerly Trinean DropSense16); devices without content profiling that differentiates turbidity may give lower values
+++ Size selection is recommended.


 

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