1156 p8H-VentExo
Cloning of p8H-VentExo
[edit]Purpose: Cloning of p8H-VentExo
Date: 2012-06-13
Notebook:
Page:
Clone ID: 1156
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]Date: June 13, 2012
Ran PCR using 423 pET30b-DeepVent-sso7d as template and VentFire-Nde1-F and VentFire-Xho1-R as primers. Used KAPA enzyme with 20 cycles and 45 seconds extension time.
Ran PCR gel, product is supposed to be ~1.3 kb, see lanes 9 and 10
Cut out bands (lane 9 and 10) , purified DNA and digested DNA and vector p8H (1135) with restriction enzymes Xho1 and Nde1
Lane 6 is the DNA digestion and lane 7 is the p8H vector digestion. Cut out bands, purify and ligate overnight.
Date: June, 14 2012
Tranformed cells with DH5 alpha cells in the morning, plated on KAN plates
Date: June 22, 2012
Cells came with with a mutation, might have had a tail to tail join. Started all over
Cut out band in lane 1 (expected size 1,365 bp) , digested with p8H (1135) with restriction enzymes Nde1 and Xho1
Cut out bands in lanes 2 and 3, prepared for ligation
May have accidentally switched samples, reamplified sequence
No band in lane 1, need to reamplify on Monday, expected size is 1.4 kb.
Date: July 10, 2012
Cut out band in lane 1 (expected size 1,365 bp) , digested with p8H (1135) with restriction enzymes Nde1 and Xho1
Digestion of DNA did not appear (lane 2), saved digested vector (lane 2), reamplify and digest tomorrow
Date: July 11, 2012
Reamplified DNA from 423 pET30b-DeepVent-sso7d as template and VentFire-Nde1-F and VentFire-Xho1-R as primers, used PHUSION enzyme 25 cycles (see lanes 1 and 2)
Cut out bands, purified and digested with restriction enzymes Nde1 and Xho1
Digested DNA with Nde1 and Xho1, ligated it together with digested vector p8H (1135) from yesterday
Ligate over night, transform with Top10 cells and plate on KAN plates tomorrow morning
Colony Screening
[edit]Date: June 15, 2012
Colonies grew on plates, screened 7 samples for PCR screening
All samples positive, grew 1, 2, 4, 5, 6 and 7 is KAN media overnight
Date: June 18, 2012
Did Miniprep, purified plasmids, digested with Nde1 and Xho1 to confirm positive clones
Size of band is a little too big. Digested with different enzymes to check for fragment sizes.
DNA ladder 5000, 3000, 2000, 1500, 1000, 750, 500, 300, 100
| Lane |
Sample |
Enzymes |
| 1 |
1 |
Xho1 + Kpn1 |
| 2 |
1 |
Xho1 + Kpn1 |
| 3 |
2 |
HindIII + Xho1 |
| 4 |
2 |
HindIII + Xho1 |
Submitted sample one for sequencing
Date: June 21, 2012
Sequencing came back with tail to tail, redigest sample 1 to check for the length of the plasmid
Redigested 1156 to check for size. Looks like it is a tail to tail join
Digested 5 uL of sample 2 with HindIII and Kpn1 separately to check
| Lane | Sample | Enzyme |
| 1 | 2 | HindIII |
| 2 | 2 | KpnI |
Date: July 16, 2012
Digested plasmids with Nde1 and Xho1, expected size 1.3 kb. All samples postive, sent sample 1 to sequencing (lane 1)
Date: July 17, 2012
Band is too high (see lane 2) Digested plasmid 1 with Nde1 and Xho1, expected size 1.3 kb
Date: July 19, 2012
Digested plasmids with Nde1 and Xho1, submitted sample 1 to sequencing
Result intepretation/Summary
[edit]Conclusion
[edit]