Colony PCR of pcDNA3.1(+)-LovTAP (part 2)

8 colonies of each of the 3 ligation plates and 1 of the negative control plate had been dipped into Lyse in Go and then used as PCR template (1ul/20ul reaction).

File:Team-EPF-Lausanne-ladder.JPG

Comments

We can notice a certain weird consistency in the results, though the general tendency here is not what we expected ! Only colonies C4, C5 and C6 show promise.

Ligation of purified PCR products into backbones

PCR products have been purified the day before.

For Fussenegger:

• TNFR into pGL
• eGFP into pGL

SEAP can't be ligated yet! Digestion of pGL with MfeI required!

For LovTAP:

• Matt's PCR LovTAP into pMP
• RO into pcDNA3.1+

Comments

Insert comments about what happened.

VP16 Activity check

I. VP16 sample preparation 1. VP16 has been aliquoted and stocked in -80 degree again. (There are Good and Bad labeled VP16 - Good means it has been preserved in -80 and Bad means it once had been in room temperature for a while so maybe denatured) 2. Diluted the VP16 sample with 1x TBST solution and made 10microG/microL of VP16 sample. 3. Loaded 5microG / 10microG / 15microG of VP16 for Good and Bad each thus 6 samples.

II. CHO cell mixture with VP16 1. CHO cell has been lysated and mixed with VP16. 2. Made some gradient level of CHO cell concentration - 20 / 30 / 40 microL with Good VP16 sample.

From lane 1 - 10,

• 1. Ladder = 7 microL
• 2. 20microL of CHO cell lysis + 1microL of VP16 (10microG) + 29microL of SDS lysis buffer = 50microL total
• 3. 30microL of CHO cell lysis + 1microL of VP16 (10microG) + 19microL of SDS lysis buffer = 50microL total
• 4. 40microL of CHO cell lysis + 1microL of VP16 (10microG) + 9microL of SDS lysis buffer = 50microL total
• 5. 24.5microL of SDS lysis buffer + 0.5microL of VP16 (5microG) = 25microL total (Good)
• 6. 24microL of SDS lysis buffer + 1microL of VP16 (10microG) = 25microL total (Good)
• 7. 23.5microL of SDS lysis buffer + 1.5microL of VP16 (15microG) = 25microL total (Good)
• 8. 24.5microL of SDS lysis buffer + 0.5microL of VP16 (5microG) = 25microL total (Bad)
• 9. 24microL of SDS lysis buffer + 1microL of VP16 (10microG) = 25microL total (Bad)
• 10. 23.5microL of SDS lysis buffer + 1.5microL of VP16 (15microG) = 25microL total (Bad)

I stocked this in -4' refrigerator and I will tag antibodies on Monday.