Team:UNIPV-Pavia/Calendar/September/settimana3
From 2010.igem.org
m (→September, 17th) |
(→September, 16th) |
||
(24 intermediate revisions not shown) | |||
Line 65: | Line 65: | ||
---- | ---- | ||
- | I52, I54, I55, I56, I57, I58 plates showed in general few colonies; I52 and | + | I52, I54, I55, I56, I57, I58 plates showed in general few colonies; I52 and I55 showed very few colonies (<=5). Very strange (mumble mumble...), they will be screened ASAP (stored at +4°C). |
Miniprep and quanfification of: | Miniprep and quanfification of: | ||
Line 88: | Line 88: | ||
and repeat | and repeat | ||
*I53: I32 (E-S) + I37 (E-X) | *I53: I32 (E-S) + I37 (E-X) | ||
- | |||
- | |||
- | |||
---- | ---- | ||
Line 101: | Line 98: | ||
Negative controls were ok and maybe MC42_C and MC43_C were ok too but further investigations were necessary. | Negative controls were ok and maybe MC42_C and MC43_C were ok too but further investigations were necessary. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | 5 ul of MG42 and MG43 were streaked on LB+Cm12.5 agar plates and incubated ON at 43°C. | ||
---- | ---- | ||
Line 109: | Line 110: | ||
==September, 15th== | ==September, 15th== | ||
- | |||
- | |||
- | |||
Transformation of I 53, I59, I60, I61 ligations into ''E. coli'' DH5-alpha. They were plated on LB+Amp agar plates and let grow ON at 37°C. | Transformation of I 53, I59, I60, I61 ligations into ''E. coli'' DH5-alpha. They were plated on LB+Amp agar plates and let grow ON at 37°C. | ||
---- | ---- | ||
+ | 3 colonies were picked from each MG plates and screening PCR was performed also with MC42 and MC43 samples. Two methods with different DNA polymerases were used in order to identify best experimental conditions (Taq polymerase and Pfx accuprime polymerase). The amplicons length (about 4Kb) probably was the reason of our problem in all the PCRs. | ||
+ | |||
+ | [[Image:UNIPV10_15_09_10_PCR_MARKER_MC42ABC_MG42ABC_Cneg_BLANK_MARKER_MG42ABC_MG43ABC_Cneg_BLANK(sopra Taq_sotto Pfx).jpg|thumb|250px|center| Screening PCR. In order: MC42ABC, MG42ABC, Cneg, BLANK, MG42ABC, MG43ABC, Cneg, BLANK (Taq samples over and Pfx samples under)]] | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
Line 141: | Line 142: | ||
I58a/b/c, I59a/b/c, I60a/b/c and I61a/b/c were let grow ON at 37°C in order to perform miniprep the following day. | I58a/b/c, I59a/b/c, I60a/b/c and I61a/b/c were let grow ON at 37°C in order to perform miniprep the following day. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | MC and MG cultures OD was referred to the lowest one in order to perform Tecan Test (note that MG42-A didn't grow so we didn't perform test on this sample). Samples were then centrifuged: the resulting pellets were resuspended on 1 ml of PBS. | ||
---- | ---- | ||
Line 171: | Line 176: | ||
*I61a/b/c: E-P | *I61a/b/c: E-P | ||
Agarose gel was prepared and samples loaded and run (and cut were necessary) | Agarose gel was prepared and samples loaded and run (and cut were necessary) | ||
- | [[Image:UNIPV10_17_09_10_digestioni_screening_I58-I61.jpg|thumb|200px|center| Screening and gel extraction for positive | + | {|align="center" |
- | As you can see they are all positive. So we stocked and gel extracted I58c, I59b, I60a and only stocked I61a. | + | |[[Image:UNIPV10_17_09_10_digestioni_screening_I58.jpg|thumb|40px|center| Gel extraction for I58.]] || [[Image:UNIPV10_17_09_10_digestioni_screening_I59-I61.jpg|thumb|200px|center| Screening and gel extraction for positive I59, I60. Screening for I61.]] |
- | + | |} | |
- | + | As you can see they are all positive (I58 was run a little longer to better separate bands). So we stocked and gel extracted I58c, I59b, I60a and only stocked I61a. | |
- | + | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
Line 181: | Line 185: | ||
==September, 18th== | ==September, 18th== | ||
Gel extraction and quantification of: | Gel extraction and quantification of: | ||
- | *I58 (E-S): | + | *I58 (E-S): 13.8 ng/ul |
- | *I59 (X-P): | + | *I59 (X-P): 24.6 ng/ul |
- | *I60 (E-S): | + | *I60 (E-S): 12.8 ng/ul |
DNA was than stored at -20°C. | DNA was than stored at -20°C. | ||
- | |||
- | |||
- | |||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==September, 19th== | ==September, 19th== | ||
+ | Since there were no more colonies in I55 agar plate, we had to transform it again to perform screening through colony PCR. So I55 was transformed again into ''E. coli'' DH5-alpha and plated on LB+Amp agar plate, that was let grow ON at 37°C. | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> |
Latest revision as of 11:16, 26 October 2010
|
||||||||||||||||||||||||||||||||||
|
|