Restriction Enzymes BamHI

A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence. The four most common types of restriction enzymes inclue: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). The most common challenges with restriction digest include- 1. inactivation of enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with activity of the other. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.

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Found 5 matching solutions for this experiment

BamHI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Protocol tips
All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Protocol tips

All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Publication protocol 1 Relevant paper

BamHI NEB #R0136

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and afterward digested with HindIII and BamHI (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA Minicore (Center for Cancer Research, National Cancer Institute, National Institutes of Health).

Protocol tips
The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and afterward digested with HindIII and BamHI (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA Minicore (Center for Cancer Research, National Cancer Institute, National Institutes of Health).

Protocol tips

The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and afterward digested with HindIII and BamHI (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA Minicore (Center for Cancer Research, National Cancer Institute, National Institutes of Health).

Publication protocol 1 Relevant paper

BamHI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Each fragment was amplified with a forward primer containing a BamHI restriction site and a reverse primer with a stop codon and a HindIII restriction site. After digested with BamHI and HindIII (Takara), the PCR products were cloned into expression vector, pET-32a(+) (Novagen).

Protocol tips
Each fragment was amplified with a forward primer containing a BamHI restriction site and a reverse primer with a stop codon and a HindIII restriction site. After digested with BamHI and HindIII (Takara), the PCR products were cloned into expression vector, pET-32a(+) (Novagen).

Manufacturer protocol Publication protocol 1 Relevant paper

BamHI R6025

Promega

Upstream tips	Protocol tips	Downstream tips
	Both coding sequences were excised with HindIII and BamHI (Promega). The vector pcDNA3.1+ (Invitrogen) was digested with HindIII and BamHI. The digested components were gel-purified and ligated overnight at room temperature using T4 DNA ligase (Promega).

Protocol tips
Both coding sequences were excised with HindIII and BamHI (Promega). The vector pcDNA3.1+ (Invitrogen) was digested with HindIII and BamHI. The digested components were gel-purified and ligated overnight at room temperature using T4 DNA ligase (Promega).

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest BamHI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	For each of the three gRNA expression cassettes, set up a restriction endonuclease digestion reaction as described below. Digest separately the gRNA expression segment gRNA1 with EcoRI and BamHI, gRNA2 with BamHI and NcoI, and gRNA3 with NcoI and NotI. Incubate the digestion mixture at 37 °C for 30 min. Purify the DNA from the digestion mixture with the Promega gel and PCR clean-up system. We show digestion of gRNA1 expression cassette from Step 12 as an example. Digest gRNA2 and gRNA3 with the corresponding enzymes in the same manner.

Protocol tips
For each of the three gRNA expression cassettes, set up a restriction endonuclease digestion reaction as described below. Digest separately the gRNA expression segment gRNA1 with EcoRI and BamHI, gRNA2 with BamHI and NcoI, and gRNA3 with NcoI and NotI. Incubate the digestion mixture at 37 °C for 30 min. Purify the DNA from the digestion mixture with the Promega gel and PCR clean-up system. We show digestion of gRNA1 expression cassette from Step 12 as an example. Digest gRNA2 and gRNA3 with the corresponding enzymes in the same manner.

Protocol tips

For each of the three gRNA expression cassettes, set up a restriction endonuclease digestion reaction as described below. Digest separately the gRNA expression segment gRNA1 with EcoRI and BamHI, gRNA2 with BamHI and NcoI, and gRNA3 with NcoI and NotI. Incubate the digestion mixture at 37 °C for 30 min. Purify the DNA from the digestion mixture with the Promega gel and PCR clean-up system. We show digestion of gRNA1 expression cassette from Step 12 as an example. Digest gRNA2 and gRNA3 with the corresponding enzymes in the same manner.

Manufacturer protocol Publication protocol 1 Relevant paper

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