253 pET28b-T4 Ligase

gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp

Purpose: gene synthesis of T4 dna ligase and ligate to pet 28b and pdbhis.mbp.

Date: 2011-8-15

Notebook: Haiyan

Page:003023

Clone ID: 253

Experimenters: Haiyan

Results

Subcloning

Date: 2011-8-15

PCR amplify T4 dna ligase and Digest with NdeI + xhoi, lane 1-3

Digest pet 28b (lane 4) and pdbhis.mbp (lane 5, 6) with NdeI + xhoi

Result: T4 dna ligase no amplify bands, using fusion enzyme to amplify again.

Colony Screening

Date: 2011-8-15

Make miniprep and digest with NdeI + BamH1

Lane 1: pLVGP-PKAalpha 3+4, Xho1 + Mlu1

Lane 2: pLentiGP #1, Xho1 + Mlu1

Lane 3: pLentiGP #6, Xho1 + Mlu1

Lane 4: pLVGP #1, Xho1 + Mlu1

Lane 5: pLVGP-PKAalpha 3+4, Nde1+ BamH1

Lane 6: pLentiGP 1, Nde1+ BamH1

Lane 7: pLentiGP 6, Nde1+ BamH1

Lane 9: pLVGP1, Nde1+BamH1

Result intepretation/Summary

• PKAalpha 3+4 has one internal Xho1 site. GFP-Puro can be cut out with Xho1 + Mlu1. So Lane 1 has correct number of bands.
• PKA alpha 3+4 with CMV promoter can be cut out with Nde1+ BamH1. Lane 5 has correct number of bands.
• lane 6 maybe good (incomplete digestion)
• Sequencing Result:
  

Conclusion

Restriction digestion

shows  pLentiGP clone #1 may be good,

pLVGP-PKAalpha 3+4 is good

Paul.xu 08:23, 15 June 2011 (EST)