Difference between revisions of "IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week10/Chemical and Light"

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(Single-cut Digest of P124-5 8/27)
(Ligating mtrB TOPO with RBS)
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==Ligating mtrB TOPO with RBS==
 
==Ligating mtrB TOPO with RBS==
Grew up more mtrB TOPO for next week.
+
Grew up more mtrB TOPO for next week.  Will need to grow up more P40 and P97 next week then, too.
  
 
==PCR of mtrB with RBS + mtrB primers==
 
==PCR of mtrB with RBS + mtrB primers==
 
Scheduled for next week.
 
Scheduled for next week.

Revision as of 22:47, 30 August 2008

Transformations in Shewie

About 250 ng of DNA added each time.

First Set 8/22

Plate Colonies Size Color
S1 P102 (P108+45) Kan 5 colonies 3mm to 5 mm Pink
S1 P121+P38 (P124) 1:2.5 Kan A 1 colony 5mm to 7.5mm Pink
S1 P28 (ColE1) Kan 0 colonies
S1 P121+P38(P124) 1:2.5 Kan B 0 colonies
S1 P121+P39 1:2.5 Dephos Kan A 0 colonies
S1 Topo vector (puc) 2ul Kan X-gal >50 colonies 1mm White
S1 Topo vector (puc) 4ul Kan X-gal >50 colonies 1mm White
S1 P121+P39 1:2.5 Kan D 0 colonies
S1 (-) ctrl (just cells) Kan 0 colonies
S1 (+) ctrl (E1 p59b) Kan 2 colonies 5mm Pink

Poor transformation efficiency in the positive control suggests that there may be a problem with our cells. All samples, except for P102, were retransformed the following week with fresh cell cultures.

Second Set (with new cells) 8/27

Plate Marker Description
S1 P124 1:2.5 A Kan 14 2-3mm pink colonies, ~100 0.5mm white colonies
S1 P124 1:2.5 B Kan 2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 Dephos A Kan 2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 D Kan 8 2mm pink colonies, ~100 0.5mm white colonies
S1 P28 Kan ~8 1-2mm pink colonies, ~50 0.5mm white colonies
S1 TOPO 2uL Kan Lawn of (>200) 0.5mm white colonies
S1 TOPO 4 uL (w/ Xgal) Kan Lawn of (>200) 0.5mm white colonies
S1 P59b (+) ctrl Kan ~50 2mm pink colonies, ~50 0.5mm white colonies
S1 (-) ctrl Kan 50-100 0.5mm white colonies

Getting Thermoinducible and Lac Inducible GFP into pSC101

Digests of Inducible Systems and pSC101 8/27

P102 needs to be retransformed so it was not included in this set.

Gel of Digest 8/27

8-27-08 amy digests.jpg 1% agarose
Lane Sample
1 1 kb + DNA Ladder
2 P124 1:2.5 A
3 P124 1:2.5 B
4 P125 1:2.5 Dephos A
5 P125 1:2.5 D
6 mtrB TOPO 0.5 uL B
7 mtrB TOPO 2 uL B
8 mtrB TOPO 4-1
9 mtrB TOPO 4-10

mtrB TOPO bands were cut (2.2 kB) but P124-5 did not cut properly, suggesting that there was an extra cut site that was cutting the insert into two pieces.

Single-cut Digest of P124-5 8/27

8-27-08 singlecut al.jpg 1.2% E-gel visualized using EtBr/UV
Lane Sample
1 1 kb + DNA Ladder
2 P124 1:2.5 A with XbaI
3 P124 1:2.5 B with XbaI
4 P125 1:2.5 Dephos A with XbaI
5 P125 1:2.5 D with XbaI
7 P124 1:2.5 A with PstI
8 P124 1:2.5 B with PstI
9 P125 1:2.5 Dephos A with PstI
10 P125 1:2.5 D with PstI
11 1 kb + DNA Ladder

IMPORTANT NOTE Further inspection of the sequence revealed that there was an extra PstI site after the cI repressor coding region due to an error that was made while making the primers that resulted in the suffix being backward and the cut sites on the part to read EXPS. Attempts to mutate out the extra site and basepairs will begin next week once primers are reviewed and ordered-- might take a while just to get them.

Due to the extra PstI site, all future ligations involving the thermoinducible system cut with PstI will need to be held off (ie. getting into the pSC101 vector, and adding on mtrB or mtrA) until the issue is resolved either with site directed mutagenesis or by redesigning primers for cI and starting over?

Getting mtrB onto thermo and lac inducible systems

Ligating mtrB TOPO with RBS

Grew up more mtrB TOPO for next week. Will need to grow up more P40 and P97 next week then, too.

PCR of mtrB with RBS + mtrB primers

Scheduled for next week.