User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/05/06: Difference between revisions
From OpenWetWare
(Autocreate 2013/05/06 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli) |
(→Date) |
||
Line 7: | Line 7: | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
= | =06/05/2013= | ||
''' | '''Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)''' | ||
# | |||
*Ligation of construct BD001(KAH182-KAH201) and mammalian vector V0200 and cloning in Bl21-DE3. | |||
*KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121. | |||
*Assembly strategy: The backbone V0200(N/S)/4442 , the insert is KAH182-KAH201(N/S)/1901. | |||
'''Digests''' <br> | |||
[[Image:KAH182-KAH201-V0200-S-N Digestion.jpg|thumb|350px| (1) KAH182-KAH201, 1901 bp, (2) V0200, 4442 bp, Both 1 and 2 are cut with Spe1 and Not11]] | |||
{| {{table}} cellspacing="3" width=350px | |||
| Plasmid DNA || 15.00 μl | |||
|- | |||
| NotI || 1.0 μl | |||
|- | |||
| SpeI || 1.0 μl | |||
|- | |||
| 10x FastDigest buffer + green loading dye || 3.00 μl | |||
|- | |||
| dH<sub>2</sub>O || 10.00 μl | |||
|- | |||
| || 30.0 μl total | |||
|- | |||
| Incubate at 37°C for 10 minutes. | |||
|} | |||
* '''Ligations''' | |||
{| {{table}} cellspacing="3" <!-- Ligations table --> | |||
|- bgcolor=#cfcfcf | |||
| Ligation || <font color="blue"><u> | |||
|- | |||
| 1. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 3:1 Six Colonies</font> | |||
|- | |||
| 2. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 4:1 One Colony</font> | |||
|- | |||
| 3. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 5:1 One Colony</font> | |||
|- | |||
| 4. E. coli BL21 KAH182-KAH201(N/S)/size, 21.00 ng + V0200(N/S)/size, 43.00ng || <font color="blue">KAH182/KAH201 10:1 One Colony</font> | |||
|- | |||
| 5. KAH201(S/P)/size, 36.29ng (Control Plate)|| <font color="blue">No Colony</font> | |||
|} | |||
* Calculations are for the ng of insert we need to get a 3:1, 4:1, 5:1 and 10:1 ratios of insert molecules to 20 ng vector molecules | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 (3:1) || 2 (4:1) || 3 (5:1) ||4 (10:1) || 5 (- Ctrl) | |||
|- | |||
| Insert DNA || 1.22 || 1.63 || 2.04 || 4.08 || --- | |||
|- | |||
| Vector DNA || 1.62 || 1.62 || 1.62 || 1.62 || 1.62 | |||
|- | |||
| 2x lgn buf (Roche) || 5.00 || 5.00 || 5.00 || 5.70 || 5.00 | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 1.16 || 0.75 || 0.34 || --- || 2.38 | |||
|- | |||
| || 10.00 μL || 10.00 μL || 10.00μl || 12.40μL || 10μL | |||
|} | |||
* The incubation time for the ligation process was 15min at room temperature. | |||
* Fast transformation, 15 min on ice. |
Revision as of 12:59, 6 May 2013
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
06/05/2013Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)
|