Additional Materials for Cloning

For Digestion of Uncut Cloning Vector

• Restriction enzymes (Recommended supplier: New England BioLabs)

For Ligating and Transforming cDNA Constructs

• Cellecta cDNA or InDOXible Cloning Vector
• T4 DNA Ligase and 10X Ligation Reaction Buffer (Recommended: New England BioLabs, T4 DNA Ligase, 400 U/μl. Cat.# M0202S): Before using, dilute T4 DNA Ligase 10-fold with 1X T4 DNA ligase buffer to 40 U/μl.)
• Competent E. coli cells (RecA-) (Recommended: Invitrogen, OmniMAX 2 T1 Phage-resistant cells, Cat.# C8540-03)
• Petri plates containing LB Agar media with 100 μg/ml Ampicillin or Carbenicillin

For Screening cDNA Inserts

• Taq DNA polymerase, and 10X reaction buffer (Recommended: Takara Clontech Titanium Taq DNA polymerase, Cat.# 639208)
• dNTP mix (Recommended: GE/Amersham, dNTP set, Cat. # 27-2035-01)
• Thermal Cycler
• 3% 1X TAE Agarose gel
• Insert Screening PCR Primers for your vector

For Purifying Constructs after Cloning

• Plasmid purification kit (Recommended: QIAGEN Endotoxin-free Plasmid Kit. The following kit combinations can be used for Midi scale preparation of endotoxin-free DNA:
  • QIAfilter Plasmid Midi Kit, Cat.# 12243, and EndoFree Plasmid Maxi Kit, Cat.# 12362
  • QIAfilter Plasmid Midi Kit, Cat.# 12243, and EndoFree Plasmid Buffer Set, Cat.# 19048