Molecular Biology Protocols

 
Marine DNA Sequencing and Analysis Center
Mount Desert Island Biological Laboratory

Last update February 5, 2009
Send comments to dtowle@mdibl.org

List of Materials
Protocol 1:  Primer Design

Internet access

Lasergene and Primer Premier software

 

Protocol 2:  DNA Isolation

Qiagen DNeasy Tissue Kit (Cat. No. 69504, qiagen.com)

Heating block for 55 and 70oC

Vortexer

Balance for weighing milligram amounts of tissue

Dissection instruments

P200 and P20 micropipettors and presterilized filter tips

1.5-ml microcentrifuge tubes, nuclease-free

Microcentrifuge

Ethanol (96-100%)

 

Protocol 3A:  RNA Isolation: Phenol-Based Procedure

RNAseZap (ambion.com)

RNAgents Total RNA Isolation System (Cat. No. Z5110, promega.com)

Ethanol (Cat. No. E702-3, sigmaaldrich.com)

Nuclease free water (Cat. No. P1193, promega.com)

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

Dissecting tools

Sterile polypropylene centrifuge tubes (12-ml for up to 250 mg tissue) (Cat. No. 718-PG, dotscientific.com)

Microcentrifuge tubes (Cat. No. 819-S, dotscientific.com)

Sterile 5- or 10-ml glass pipets and pipet controller

P1000 micropipettor and presterilized filter pipet tips (Rainin GP-1000F)

Homogenizer and probe:  Omni TH with 7 x 195 mm sawtooth probe (omni-inc.com) or equivalent

Eppendorf Refrigerated Centrifuge (Model 5810R) and rotors

Vortexer

Non-frost-free freezer (-20C)

 

Protocol 3B: RNA Isolation: Filter-Based Procedure

RNAlater (ambion.com)

RNAseZap (ambion.com)

SV Total RNA Isolation System (Cat. No. Z3102, promega.com)

Ethanol (Cat. No. E702-3, sigmaaldrich.com)

Nuclease free water (Cat. No. P1193, promega.com)

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

Dissecting tools

Sterile polypropylene centrifuge tubes (12-ml for up to 350 mg tissue) (Cat. No. 718-PG, dotscientific.com)

Microcentrifuge tubes (Cat. No. 819-S, dotscientific.com)

P1000 micropipettor and presterilized filter pipet tips (Rainin GP-1000F)

Homogenizer and probe:  Omni TH with 7 x 195 mm sawtooth probe (omni-inc.com) or equivalent

Microcentrifuge (Eppendorf Mini-Spin, for example)

Non-frost-free freezer (-20C)

 

Protocol 4:  RNA Analysis

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

RNA 6000 Nano LabChip Kit (Cat. No. 5067-1511, agilent.com)

RNAseZAP (Cat. No. 9780, ambion.com)

P1000 and L2 micropipettors and presterilized filter pipet tips (RT-1000F and RT-L10F, rainin.com)

Microcentrifuge tubes (0.6-ml and 1.7-ml) (Cat. No. 502-PCR and RN-1700-GMT, dotscientific.com)

Microcentrifuge (Eppendorf 5417 or equivalent)

Agilent 2100 Bioanalzyer

 

Protocol 5:  Reverse Transcription

SuperScript III First-Strand Synthesis System (Cat. No. 18080-051, invitrogen.com)

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

P1000, P200, P20, and L2 micropipettors and presterilized filter pipet tips (rainin.com RT-1000F, HR-100F, RT-10GF, RT-L10F)

Microcentrifuge tubes (0.2-ml and 1.7-ml) (Cat. No. 201-PCR and RN-1700-GMT, dotscientific.com, or equivalent)

Thermal cycler for 0.2-ml tubes (MJ Research PTC200 or equivalent)

Microcentrifuge (Eppendorf 5417 or equivalent)

Tube rack

 

Protocol 6:  PCR

Primers from IDT http://xenopus.mdibl.org/orders/oligo.html

Tris-EDTA (TE) buffer (Cat. No. T9285, sigmaaldrich.com)

PCR Core Kit (RedTaq ReadyMix, Cat. No. R2523, sigmaaldrich.com)

Nuclease-free water (Cat. No. P1193, promega.com)

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

P1000, P200, P20, and L2 micropipettors and presterilized filter pipet tips (rainin.com RT-1000F, HR-100F, RT-10GF, RT-L10F)

Microcentrifuge tubes (0.2-ml and 1.7-ml) (Cat. No. 201-PCR and RN-1700-GMT, dotscientific.com)

Thermal cycler for 0.2-ml tubes (MJ Research PTC200 or equivalent)

Microcentrifuge (Eppendorf 5417 or equivalent)

Tube rack

 

Protocol 7: Gel Electrophoresis

Mini-gel apparatus with combs (MiniSubCell GT, biorad.com, or equivalent)

Power supply (PowerPac 200, biorad.com, or equivalent)

Agarose (Cat. No. V3121, promega.com)

Tris-Borate-EDTA (TBE) buffer (Cat. No. V4251, promega.com)

DNA ladder (Cat. No. 10068-013, invitrogen.com)

Loading dye (Cat. No. G1881, promega.com)

SYBR Safe DNA Gel Stain (Cat. No. S33101, invitrogen.com)

Safe Imager Blue-Light Transilluminator (invitrogen.com)

Digital imaging system

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

P20 micropipettors and presterilized filter pipet tips (rainin.com HR-100F, RT-10GF)

Microcentrifuge tubes (0.6-ml) (Cat. No. 502-PCR, dotscientific.com)

Tube rack

 

Protocol 8:  Gel Extraction

MinElute Gel Extraction Kit (Cat. No. 28604, qiagen.com)

Scalpels (Cat. No. GFI2975-20, daigger.com)

Ethanol (Cat. No. E702-3, sigmaaldrich.com)

Safe Imager Blue-Light Transilluminator (invitrogen.com)

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

P1000 micropipettors and presterilized filter pipet tips (rainin.com RT-1000F)

Microcentrifuge tubes (1.7-ml) (Cat. No. RN-1700-GMT, dotscientific.com)

Hot block or water bath at 50C

Microcentrifuge (Eppendorf 5417 or equivalent)

Tube rack

 

Protocol 9:  Preparing for Sequencing

Materials for Protocol 7

Sequencing primers

 

Protocol 10:  Analysis of DNA Sequences

Internet access

Lasergene software

 

Protocol 11:  Quantitative PCR

SYBR Green QPCR Master Mix, Brilliant II (Cat. No. 600828, stratagene.com)

Strip tubes and caps for MX3005 instrument (Cat. Nos. 401428 and 401425, stratagene.com)  DO NOT USE SUBSTITUTES!

Primers

Nuclease-free water (Cat. No. P1193, promega.com)

Disposable gloves (Cat. No. 1802-PF and others, dotscientific.com)

P1000, P200, P20, and L2 micropipettors and presterilized filter pipet tips (rainin.com RT-1000F, HR-100F, RT-10GF, RT-L10F)

Quantitative PCR System (Stratagene MX3005 or equivalent)